PC5 as a Factor IX Propeptide Processing Enzyme

ABSTRACT

Compositions and methods for preparing Factor IX, Factor IX-containing fusion proteins, and Factor IX-containing conjugates with processing of Factor IX propeptide by PCS, are provided. In one embodiment PCS is used to process a precursor polypeptide for a Factor IX-Fc monomer-dimer hybrid.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.60/785,421, filed Mar. 24, 2006, the entire content of which isincorporated herein by reference.

BACKGROUND OF THE INVENTION

Factor IX (FIX) is a single-chain, 55 kDa zymogen of a serine proteaseencoded on the X chromosome in humans that is an important component ofthe intrinsic pathway of the blood coagulation cascade. Deficiency offunctional FIX causes hemophilia B, also known as Christmas disease.Hemophilia B is reported to occur in 1 in 100,000 male births and, whenuntreated, is associated with severe and chronic morbidity resultingfrom uncontrolled bleeding into muscles, joints, and body cavitiesfollowing injury. Until recently, treatments for FIX deficiency haveincluded administration of natural FIX prepared from plasma derived fromblood donor pools. Such treatments carry attendant risks of infectionwith blood-borne viruses including human immunodeficiency virus (HIV)and hepatitis C virus (HCV), as well as unwanted thrombosis andembolism. More recently, a preparation of recombinant FIX (BeneFIX®,Wyeth) became commercially available.

Certain posttranslational modifications are required for normal FIXactivity. Fix is expressed as a precursor polypeptide that requiresposttranslational processing to yield mature FIX. In particular, theprecursor polypeptide of FIX requires vitamin K-dependent gammacarboxylation of certain glutamic acid residues in the so-calledgamma-carboxyglutamate (Gla) domain and cleavage of propeptide (see FIG.1). The propeptide is an 18-amino acid residue sequence N-terminal tothe Gla domain. The propeptide binds vitamin K-dependent gammacarboxylase and then, in vivo, is cleaved from the precursor polypeptideof FIX by an endogenous protease, most likely PACE (paired basic aminoacid cleaving enzyme), also known as furin and PCSK3. Without thevitamin K-dependent gamma carboxylation, the Gla domain is unable tobind calcium to assume the correct conformation necessary to anchor theprotein to negatively charged phospholipid surfaces, thereby renderingFactor IX nonfunctional. Inhibition of vitamin K-dependent carboxylationby vitamin K antagonists such as warfarin is a common form ofanticoagulant therapy. Even if it is carboxylated, the Gla domain alsodepends on cleavage of the propeptide for proper function, sinceretained propeptide interferes with conformational changes of the Gladomain necessary for optimal binding to calcium and phospholipid. Thusrequired post-translational modifications of the precursor polypeptideof FIX include both gamma carboxylation of certain glutamic acidresidues by vitamin K-dependent gamma carboxylase and cleavage of theFIX propeptide, most likely by PACE, to yield mature FIX.

Mature FIX must be activated by activated Factor XI to yield Factor IXa.In the intrinsic pathway, FIX associates with a complex with activatedFactor VIII, Factor X, calcium, and phospholipid, wherein FIX isactivated by Factor XIa, and then Factor IXa in turn activates Factor Xin concert with activated Factor VIII. Alternatively, Factors IX and Xcan both be activated by Factor VIIa complexed with lipidated TissueFactor, which has been generated via the extrinsic pathway. Factor Xathen participates in the final common pathway whereby prothrombin isconverted to thrombin, which in turn converts fibrinogen to fibrin.

Until now, in vitro post-translational processing of the precursorpolypeptide of FIX, consistent with what was known about in vivoprocessing, has relied on PACE to effect cleavage of FIX propeptide.PACE is a member of a family of at least a half dozen mammaliansubtilisin/Kex2p-like serine proteases known as proprotein convertases(PCs). PACE was found using sequence homology to KEX2, an enzyme in theyeast Saccharomyces cerevisiae and the first to be identified as anendoprotease involved in precursor processing. Subsequently other PCfamily members have been identified and found to have varying degrees ofsequence identity and different substrate specificities.

EP 0246709 describes partial cDNA and amino acid sequences of furin(i.e., PACE).

Complete cDNA and amino acid sequences of human furin (i.e., PACE) werepublished in 1990. Van den Ouweland A M et al. (1990) Nucleic Acids Res.18:664; Erratum in: Nucleic Acids Res. 18:1332 (1990).

U.S. Pat. No. 5,460,950, issued to Barr et al., describes recombinantPACE and the coexpression of PACE with a substrate precursor polypeptideof a heterologous protein to improve expression of active, matureheterologous protein. In one embodiment the precursor polypeptide is aprecursor polypeptide of FIX.

U.S. Pat. No. 5,935,815, issued to van de Ven et al., likewise describesrecombinant human furin (i.e., PACE) and the coexpression of furin witha substrate precursor polypeptide of a heterologous protein to improveexpression of active, mature heterologous protein. Possible substrateprecursors disclosed in this patent include a precursor of Factor IX.

Other family members in the mammalian subtilisin/Kex2p-like proproteinconvertase (PC) family in addition to PACE are reported to includePC1/PC3, PC2, PC4, PC5/6 (hereinafter referred to simply as PC5), PACE4,and LPC/PC7/PC8/SPC7. While these various members share certainconserved overall structural features, they differ in their tissuedistribution, subcellular localization, cleavage specificities, andpreferred substrates. For a review, see Nakayama K (1997) Biochem J.327:625-35. Similar to PACE, these proprotein convertases generallyinclude, beginning from the amino terminus, a signal peptide, apropeptide (that may be autocatalytically cleaved), a subtilisin-likecatalytic domain characterized by Asp, His, Ser, and Asn/Asp residues,and a Homo B domain that is also essential for catalytic activity andcharacterized by an Arg-Gly-Asp (RGD) sequence. PACE, PACE4, and PC5also include a Cys-rich domain, the function of which is unknown. Inaddition, PC5 has isoforms with and without a transmembrane domain;these different isoforms are known as PC5B and PC5A, respectively.Comparison between the amino acid sequence of the catalytic domain ofPACE and the amino acid sequences of the catalytic domains of othermembers of this family of proprotein convertases reveals the followingdegrees of identity: 70 percent for PC4; 65 percent for PACE4 and PC5;61 percent for PC1/PC3; 54 percent for PC2; and 51 percent forLPC/PC7/PC8/SPC7. Nakayama K (1997) Biochem J. 327:625-35.

PACE and PACE4 have been reported to have partially overlapping butdistinct substrates. In particular, PACE4, in striking contrast to PACE,has been reported to be incapable of processing the precursorpolypeptide of FIX. Wasley L C et al. (1993) J Biol. Chem. 268:8458-65;Rehemtulla A et al. (1993) Biochemistry. 32:11586-90.

U.S. Pat. No. 5,840,529, issued to Seidah et al., discloses nucleotideand amino acid sequences for human PC7 and the notable ability of PC7,as compared to other PC family members, to cleave HIV gp160 to gp120 andgp41.

Nucleotide and amino acid sequences of rodent PC5 were first describedas PC5 by Lusson J et al. (1993) Proc Natl Acad Sci USA 90:6691-5 and asPC6 by Nakagawa T et al. (1993) J Biochem (Tokyo) 113:132-5.

U.S. Pat. No. 6,380,171, issued to Day et al., discloses nucleotide andamino acid sequences for human PC5A, the isoform without thetransmembrane domain, as well as methods for reducing restenosis byusing antisense nucleic acids to inhibit PC5 activity.

SUMMARY OF THE INVENTION

The invention is based in part on the discovery by the inventors thatPC5 is effective for processing propeptide from the precursorpolypeptide of FIX. This discovery was unexpected because, as describedin detail below, closely related PC family members are completely orsubstantially incapable of processing propeptide from the precursorpolypeptide of FIX. The invention relates to methods and compositionsuseful for preparing mature Factor IX, using PC5 to process theprecursor polypeptide of FIX. The methods and compositions relate to FIXper se as well as to FIX-containing polypeptides and conjugates.

In one aspect the invention is a eukaryotic cell including a firstexpression vector encoding a proprotein of Factor IX (proFIX), or afusion protein thereof, and a second expression vector encoding afunctional PC5.

In one embodiment according to this and other aspects of the invention,the functional PC5 includes an amino acid sequence provided by SEQ IDNO:1, corresponding to PC5A, an isoform of PC5 without a transmembranedomain.

In one embodiment according to this and other aspects of the invention,the functional PC5 comprises an amino acid sequence corresponding toPC5B, an isoform of PC5 with a transmembrane domain.

In one embodiment according to this and other aspects of the invention,the first expression vector encodes proFIX.

In one embodiment according to this and other aspects of the invention,the fusion protein is a proFIX-FcRn binding partner fusion protein.

In one embodiment according to this and other aspects of the invention,the proFIX-FcRn binding partner fusion protein comprises a linkerconnecting proFIX to FcRn binding partner.

In one embodiment according to this and other aspects of the invention,the proFIX-FcRn binding partner fusion protein is a proFIX-Fc fusionprotein.

In one embodiment according to this and other aspects of the invention,the proFIX-Fc fusion protein comprises a human Fc gamma.

In one embodiment according to this and other aspects of the invention,the proFIX-FcRn binding partner fusion protein comprises a linkerconnecting proFIX to Fc.

In one embodiment according to this and other aspects of the invention,the proFIX-Fc fusion protein is a proFIX-Fc homodimer (also referred toherein simply as “dimer”).

In one embodiment according to this and other aspects of the invention,the proFIX-Fc fusion protein is a proFIX-Fc monomer-dimer hybrid (alsoreferred to herein simply as “monomer”).

In one embodiment according to this and other aspects of the invention,the fusion protein is a proFIX-albumin fusion protein.

In one embodiment according to this and other aspects of the invention,the proFIX-albumin fusion protein includes a linker connecting proFIX toalbumin.

In one embodiment according to this and other aspects of the invention,the fusion protein is a proFIX-transferrin fusion protein.

In one embodiment according to this and other aspects of the invention,the proFIX-transferrin fusion protein includes a linker connectingproFIX to transferrin.

In one embodiment according to this and other aspects of the invention,the linker is GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:16).

In one embodiment the eukaryotic cell is a mammalian cell.

In one embodiment the eukaryotic cell is a HEK 293 cell.

In one embodiment the eukaryotic cell is a CHO cell.

In one embodiment the eukaryotic cell is a BHK cell.

In one embodiment the expression vector encoding the proFIX or thefusion protein thereof, and the expression vector encoding thefunctional PC5, are a single expression vector.

The invention in one aspect is an expression vector including a firstpolynucleotide sequence encoding a proprotein of Factor IX (proFIX), ora fusion protein thereof, operably linked to an expression controlsequence permitting expression of the proFIX or the fusion proteinthereof, and a second polynucleotide sequence encoding a functional PC5,operably linked to an expression control sequence permitting expressionof the functional PC5.

In one aspect the invention is a method for producing a mature FactorIX-containing polypeptide from a proprotein of Factor IX (proFIX), or afusion protein thereof. The method according to this aspect of theinvention includes the steps of culturing a eukaryotic cell including afirst expression vector encoding a proprotein of Factor IX (proFIX), ora fusion protein thereof, and a second expression vector encoding afunctional PC5, under conditions that allow expression of both theproFIX or the fusion protein thereof and the functional PC5, andprocessing of the proFIX or the fusion protein thereof by the functionalPC5. In one embodiment the mature Factor IX-containing polypeptide is aFIX-Fc monomer-dimer hybrid. Also provided is a FIX-Fc monomer-dimerhybrid produced according to the method of this aspect of the invention.

In one aspect the invention is a method for increasing yield of a matureFactor IX-containing polypeptide from a proprotein of Factor IX(proFIX), or a fusion protein thereof. The method according to thisaspect of the invention includes the steps of culturing a eukaryoticcell including a first expression vector encoding a proprotein of FactorIX (proFIX), or a fusion protein thereof, and a second expression vectorencoding a functional PC5, under conditions that allow (a) expression ofboth the proFIX or the fusion protein thereof and the functional PC5,and (b) processing of the proFIX or the fusion protein thereof by thefunctional PC5, wherein yield of mature Factor IX-containing polypeptideis increased compared to yield of mature Factor IX-containingpolypeptide produced under similar conditions without the processing bythe functional PC5.

In one aspect the invention is a method for producing a mature FactorIX-containing polypeptide from a proprotein of Factor IX (proFIX), or aconjugate thereof. The method according to this aspect of the inventionincludes the step of contacting the proFIX or the conjugate thereof withan effective amount of functional PC5.

In one embodiment the proFIX or the conjugate thereof is proFIX.

In one embodiment the mature Factor IX-containing polypeptide is aFIX-Fc monomer-dimer hybrid. Also provided is a FIX-Fc monomer-dimerhybrid produced according to the method of this aspect of the invention.

In one embodiment the proFIX or the conjugate thereof is a PEGylatedproFIX.

In one embodiment the proFIX or the conjugate thereof is a proFIX-FcRnbinding partner fusion protein.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

The figures are illustrative only and are not required for enablement ofthe invention disclosed herein.

FIG. 1 is a schematic drawing of prepro Factor IX, including thefollowing domains: prepeptide (PRE), propeptide (PRO), Gla domain (GLA),H domain (H), EGF-B and EGF-A domains, activation peptide, and catalyticdomain. Mature Factor IX lacks the prepeptide and propeptide domains.Mature, activated Factor IX (Factor IXa) further lacks the activationpeptide, and the catalytic domain remains associated with the EGF-Adomain through a disulfide linkage.

FIG. 2A is a schematic drawing of the structure of a FIX-Fc homodimer.

FIG. 2B is a schematic drawing of the structure of a FIX-Fcmonomer-dimer hybrid.

FIG. 3 is a schematic drawing of PACE, PACE4, PC7, and PCSA (top tobottom, respectively), showing the following domains: signal peptide,propeptide, subtilisin-like catalytic domain, homo B domain, Cys-richdomain, and (PACE and PC7 only) transmembrane domain. Shown percentagesindicate percent amino acid identity with PACE within each correspondingdomain.

FIG. 4 shows FIX and FIX propeptide Western blots (reducing SDS-PAGE) ofprotein A immunoprecipitations of FIX-Fc from transiently transfectedChinese hamster ovary (CHO) cells, alone or with PACE-SOL, PC7-SOL, PC5,or KEX2-SOL.

FIG. 5 shows FIX and FIX propeptide Western blots and the correspondingSDS-PAGE gel (all under non-reducing conditions) of protein A purifiedproteins from CHO cells stably transfected with FIX-Fc, Fc, and eitherPACE-SOL or PC7-SOL. MWM, molecular weight marker.

FIG. 6 shows FIX and FIX propeptide Western blots (all undernon-reducing conditions) of protein A immunoprecipitations from CHOcells stably transfected with FIX-Fc, Fc, and PC5 (lanes 1-3), or CHOcells stably transfected with FIX-Fc either alone (lane 4) or withPACE-SOL (lane 5).

FIG. 7 shows FIX and FIX propeptide Western blots of protein Aimmunoprecipitations from HEK 293 cells transiently transfected withFIX-Fc and Fc, either without processing enzyme (lanes 3, “−”) or withPC5 (lanes 4-7) or PC7-SOL (lanes 8-11). Purified FIX-Fc from CHO cellstransfected with (lanes 2) or without (lanes 1) PACE-SOL were used ascontrols. Lanes 12, molecular weight markers.

FIG. 8 shows FIX (non-reducing) and FIX propeptide (reducing) Westernblots of protein A immunoprecipitations of FIX-Fc dimer, monomer-dimerhybrid (monomer), and Fc from transiently transfected HEK 293 cells,with KEX2-SOL (lanes 3 and 4). Purified FIX-Fc homodimer from CHO cellstransfected with (lane 2) or without PACE (lane 1) were used ascontrols.

FIG. 9 shows FIX and FIX propeptide Western blots and the correspondingSDS-PAGE gel (all under non-reducing conditions) of purified FIX-Fcmonomer-dimer hybrid (monomer) from HEK 293 cells stably transfectedwith FIX-Fc, Fc and PC5. Purified FIX-Fc homodimer from CHO cellstransfected with either PACE-SOL or PC7-SOL were used as controls.

FIG. 10 shows FIX and FIX propeptide Western blots (reducing conditions)of protein A immunoprecipitations of FIX-Fc dimer, monomer-dimer hybrid(monomer), and Fc from stably transfected HEK 293 cells, alone (lanes 3and 4) or with PC7-SOL (lanes 5 and 6). Purified FIX-Fc homodimer fromCHO cells transfected with (lane 2) or without (lane 1) PACE-SOL wereused as controls. Lane 7, molecular weight standards.

FIG. 11 shows peptide mapping data indicating the presence of proFIX-Fcin purified CHO-produced FIX-Fc without any processing enzyme, and theabsence of proFIX-Fc from purified HEK-produced FIX-Fc monomer-dimerhybrid (monomer) with PC5. TVFLDHENANKILNRPKR, SEQ ID NO:17; YGIYTK, SEQID NO:19.

FIG. 12 shows peptide mapping data indicating the presence of proFIX-Fcin purified CHO-produced FIX-Fc with PC7-SOL, and the absence ofproFIX-Fc from purified HEK-produced FIX-Fc monomer-dimer hybrid(monomer) with PC5. TVFLDHENANKILNRPKR, SEQ ID NO:17; YGIYTK, SEQ IDNO:19.

FIG. 13 shows peptide mapping data indicating the absence of propeptidefrom purified HEK-produced FIX-Fc monomer-dimer hybrid (monomer) withPC5 and from CHO-produced Factor IX alone. TVFLDHENANKILNRPKR, SEQ IDNO:17; YGIYTK, SEQ ID NO:19.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates at least in part to compositions and methodsrelated to PC5 useful for preparation of FIX and conjugates of FIX,including FIX fusion proteins. In certain particular embodiments the PC5is PC5A and the FIX fusion protein is a FIX-Fc monomer-dimer hybrid,discussed below.

The invention in one aspect relates to a eukaryotic cell that includes afirst expression vector encoding a proprotein of Factor IX and a secondexpression vector encoding a functional PC5 polypeptide. As used herein,a functional PC5 polypeptide refers to an enzymatically activepolypeptide comprising at least a homo 13 domain and a subtilisin-likecatalytic domain of a proprotein convertase PC5. In one embodiment afunctional PC5 polypeptide refers to an enzymatically active polypeptidecomprising at least a homo B domain and a subtilisin-like catalyticdomain of a human proprotein convertase PC5. In one embodiment afunctional PC5 polypeptide refers to an enzymatically active polypeptidecomprising at least a homo B domain and a subtilisin-like catalyticdomain of a proprotein convertase PC5, wherein the functional PC5 isPC5A. As mentioned above, the PC5A isomer lacks a transmembrane domain.In contrast, a PC5B isomer includes a C-terminal transmembrane domain.In one embodiment a functional PC5 polypeptide refers to artenzymatically active polypeptide comprising at least a homo B domain anda subtilisin-like catalytic domain of a proprotein convertase PC5,wherein the functional PC5 is human PC5A.

An amino acid sequence for human PC5A is provided as GenBank accessionno. NP_(—)006191 (reproduced as SEQ ID NO:1), the entire contents ofwhich are incorporated herein.

SEQ ID NO: 1MGWGSRCCCP GRLDLLCVLA LLGGCLLPVC RTRVYTNHWA VKIAGGFPEA NRIASKYGFI 60NIGQIGALKD YYHFYHSRTI KRSVISSRGT HSFISMEPKV EWIQQQVVKK RTKRDYDFSR 120AQSTYFNDPK WPSMWYMHCS DNTHPCQSDM NIEGAWKRGY TGKNIVVTIL DDGIERTHPD 180LMQNYDALAS CDVNGNDLDP MPRYDASNEN KHGTRCAGEV AAAANNSHCT VGIAFNAKIG 240GVRMLDGDVT DMVEAKSVSF NPQHVHIYSA SWGPDDDGKT VDGPAPLTRQ AFENGVRMGR 300RGLGSVFVWA SGNGGRSKDH CSCDGYTNSI YTISISSTAE SGKKPWYLEE CSSTLATTYS 360SGESYDKKII TTDLRQRCTD NHTGTSASAP MAAGIIALAL EANPFLTWRD VQHVIVRTSR 420AGHLNANDWK TNAAGFKVSH LYGFGLMDAE AMVMEAEKWT TVPRQHVCVE STDRQIKTIR 480PNSAVRSIYK ASGCSDNPNR HVNYLEHVVV RITITHPRRG DLAIYLTSPS GTRSQLLANR 540LFDHSMEGFK NWEFMTIHCW GERAAGDWVL EVYDTPSQLR NFKTPGKLKE WSLVLYGTSV 600QPYSPTNEFP KVERFRYSRV EDPTDDYGTE DYAGPCDPEC SEVGCDGPGP DHCNDCLHYY 660YKLKNNTRIC VSSCPPGHYH ADKKRCRKCA PNCESCFGSH GDQCMSCKYG YFLNEETNSC 720VTHCPDGSYQ DTKKNLCRKC SENCKTCTEF HNCTECRDGL SLQGSRCSVS CEDGRYFNGQ 780DCQPCHRFCA TCAGAGADGC INCTEGYFME DGRCVQSCSI SYYFDHSSEN GYKSCKKCDI 840SCLTCNGPGF KNCTSCPSGY LLDLGMCQMG AICKDATEES WAEGGFCMLV KKNNLCQRKV 900LQQLCCKTCT FQG 913Referring to SEQ ID NO:1, the PC5 signal peptide spans amino acidresidues 1-32, the PC5 propeptide spans amino acid residues 33-114, andthe mature PC5 protein (beginning with the subtilisin-like catalyticdomain) spans amino acid residues 115-913. In one embodiment afunctional PC5 polypeptide refers to a polypeptide having an amino acidsequence provided as SEQ ID NO:1. In one embodiment a functional PC5polypeptide refers to a polypeptide having an amino acid sequenceprovided as amino acid residues 33-913 of SEQ ID NO: 1. In oneembodiment a functional PC5 polypeptide refers to a polypeptide havingan amino acid sequence provided as amino acid residues 115-913 of SEQ IDNO:1.

Owing to the degeneracy of the genetic code, a human PC5A polypeptidecan be encoded by any suitable nucleotide sequence. In one embodiment anucleotide sequence for human PC5A is provided as nucleotides 478-3216of GenBank accession no. NM_(—)006200 (reproduced as SEQ ID NO:2), theentire contents of which is incorporated herein by reference. Thissequence encodes SEQ ID NO:1 above.

SEQ ID NO: 2 atgggctggg ggagccgctg ctgctgcccg ggacgtttggacctgctgtg cgtgctggcg ctgctcgggg gctgcctgctccccgtgtgt cggacgcgcg tctacaccaa ccactgggcagtcaaaatcg ccgggggctt cccggaggcc aaccgtatcgccagcaagta cggattcatc aacataggac agataggggccctgaaggac tactaccact tctaccatag caggacgattaaaaggtcag ttatctcgag cagagggacc cacagtttcatttcaatgga accaaaggtg gaatggatcc aacagcaagtggtaaaaaag cggacaaaga gggattatga cttcagtcgtgcccagtcta cctatttcaa tgatcccaag tggcccagcatgtggtatat gcactgcagt gacaatacac atccctgccagtctgacatg aatatcgaag gagcctggaa gagaggctacacgggaaaga acattgtggt cactatcctg gatgacggaattgagagaac ccatccagat ctgatgcaaa actacgatgctctggcaagt tgcgacgtga atgggaatga cttggacccaatgcctcgtt atgatgcaag caacgagaac aagcatgggactcgctgtgc tggagaagtg gcagccgctg caaacaattcgcactgcaca gtcggaattg ctttcaacgc caagatcggaggagtgcgaa tgctggacgg agatgtcacg gacatggttgaagcaaaatc agttagcttc aacccccagc acgtgcacatttacagcgcc agctggggcc cggatgatga tggcaagactgtggacggac cagcccccct cacccggcaa gcctttgaaaacggcgttag aatggggcgg agaggcctcg gctctgtgtttgtttgggca tctggaaatg gtggaaggag caaagaccactgctcctgtg atggctacac caacagcatc tacaccatctccatcagcag cactgcagaa agcggaaaga aaccttggtacctggaagag tgttcatcca cgctggccac aacctacagcagcggggagt cctacgataa gaaaatcatc actacagatctgaggcagcg ttgcacggac aaccacactg ggacgtcagcctcagccccc atggctgcag gcatcattgc gctggccctggaagccaatc cgtttctgac ctggagagac gtacagcatgttattgtcag gacttcccgt gcgggacatt tgaacgctaatgactggaaa accaatgctg ctggttttaa ggtgagccatctttatggat ttggactgat ggacgcagaa gccatggtgatggaggcaga gaagtggacc accgttcccc ggcagcacgtgtgtgtggag agcacagacc gacaaatcaa gacaatccgccctaacagtg cagtgcgctc catctacaaa gcttcaggctgctcggataa ccccaaccgc catgtcaact acctggagcacgtcgttgtg cgcatcacca tcacccaccc caggagaggagacctggcca tctacctgac ctcgccctct ggaactaggtctcagctttt ggccaacagg ctatttgatc actccatggaaggattcaaa aactgggagt tcatgaccat tcattgctggggagaaagag ctgctggtga ctgggtcctt gaagtttatgatactccctc tcagctaagg aactttaaga ctccaggtaaattgaaagaa tggtctttgg tcctctacgg cacctccgtgcagccatatt caccaaccaa tgaatttccg aaagtggaacggttccgcta tagccgagtt gaagacccca cagacgactatggcacagag gattatgcag gtccctgcga ccctgagtgcagtgaggttg gctgtgacgg gccaggacca gaccactgcaatgactgttt gcactactac tacaagctga aaaacaataccaggatctgt gtctccagct gcccccctgg ccactaccacgccgacaaga agcgctgcag gaagtgtgcc cccaactgtgagtcctgctt tgggagccat ggtgaccaat gcatgtcctgcaaatatgga tactttctga atgaagaaac caacagctgtgttactcact gccctgatgg gtcatatcag gataccaagaaaaatctttg ccggaaatgc agtgaaaact gcaagacatgtactgaattc cataactgta cagaatgtag ggatgggttaagcctgcagg gatcccggtg ctctgtctcc tgtgaagatggacggtattt caacggccag gactgccagc cctgccaccgcttctgcgcc acttgtgctg gggcaggagc tgatgggtgcattaactgca cagagggcta cttcatggag gatgggagatgcgtgcagag ctgtagtatc agctattact ttgaccactcttcagagaat ggatacaaat cctgcaaaaa atgtgatatcagttgtttga cgtgcaatgg cccaggattc aagaactgtacaagctgccc tagtgggtat ctcttagact taggaatgtgtcaaatggga gccatttgca aggatgcaac ggaagagtcctgggcggaag gaggcttctg tatgcttgtg aaaaagaacaatctgtgcca acggaaggtt cttcaacaac tttgctgcaa aacatgtaca tttcaaggc

The nucleotide sequence encoding the PC5 polypeptide can containsubstitutions that do not affect the amino acid sequence. In oneembodiment, the nucleotide at position 876 of GenBank accession no.NM_(—)006200 can be replaced by thymidine (T) instead of cytidine (C),but preserve the amino acid Ser 133 (corresponding to amino acidnumbering in GenBank accession no. NP_(—)006191). In one embodiment thenucleotide at position 1950 of GenBank accession no. NM_(—)006200 can bereplaced by cytidine (C) instead of thymidine (T), but preserve theamino acid Ala 491. Note that this nucleotide change at position 1950eliminates a HindIII restriction site. In one embodiment the nucleotideat position 1962 of GenBank accession no. NM_(—)006200 can be replacedby adenine (A) instead of guanosine (G), but preserve the amino acid Ser496. In one embodiment the nucleotide sequence encoding PC5 is asprovided below in Example 3. In further embodiments any combination ofthe aforementioned substitutions can be present. In yet furtheradditional embodiments similar types of substitutions are alsocontemplated by the invention and are within the skill of the skilledartisan to make and use in the practice of the invention.

The PC5 sequence can also contain other non-coding changes at thenucleotide level that can be found in the SNP database. For example, invarious embodiments Leu 16 can have an adenine (A) instead of aguanosine (G) at codon position 3; Cys 30 can have a cytosine (C)instead of a thymidine (T) at codon position 3; Thr 385 can have anadenine (A) instead of a guanosine (G) at codon position 3; Ser 495 canhave an adenine (A) instead of a guanosine (G) at codon position 3; Pro623 can have a thymidine (T) instead of a cytosine (C) at codon position3; Cys 767 can have a thymidine (T) instead of a cytosine (C) at codonposition 3; and any combination thereof. These examples are not intendedto be limiting in any way.

As used herein, a proprotein of Factor IX (proFIX) refers to any FactorIX polypeptide that includes at least a Factor IX propeptide, a Gladomain, and a Factor IX catalytic domain. The Factor IX propeptide ispositioned N-terminal adjacent to the Gla domain. In one embodiment theproprotein of Factor IX further includes a prepeptide (signal sequence)N-terminal to the propeptide. In one embodiment the proprotein of FactorIX is a proprotein of a human Factor IX. A human Factor IX proprotein inone embodiment has an amino acid sequence provided as GenBank accessionno. NP_(—)000124, reproduced below as SEQ ID NO:3. In this sequenceamino acid residues 1-28 represent a prepeptide (signal sequence); aminoacid residues 29-46 represent a propeptide; amino acid residues 47-86represent a Gla domain containing twelve glutamic acid residues; aminoacid residues 191-226 represent an activation peptide; and amino acidresidues 227-461 represent a catalytic domain. The activation peptide iscleaved by Factor XIa to yield a heavy chain and a light chain which arecovalently associated by one or more disulfide bonds. It should be notedthat in one embodiment the threonine at amino acid 194 in SEQ ID NO:3can be replaced by an alanine.

SEQ ID NO: 3MQRVNMIMAE SPGLITICLL GYLLSAECTV FLDHENANKI LNRPKRYNSG KLEEFVQGNL 60ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP 120FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR 180VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW 240QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII 300PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF 360HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE 420GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL T 461

Owing to the degeneracy of the genetic code, a proprotein of humanFactor IX can be encoded by any suitable nucleotide sequence. In oneembodiment a nucleotide sequence encoding a proprotein of human FactorIX is provided as nucleotides 30-1412 of GenBank accession no.NM_(—)000133, reproduced below as SEQ ID NO:4. This sequence encodes SEQID NO:3 above.

SEQ ID NO: 4 atgcagcgcg tgaacatgat catggcagaa tcaccaggcctcatcaccat ctgcctttta ggatatctac tcagtgctgaatgtacagtt tttcttgatc atgaaaacgc caacaaaattctgaatcggc caaagaggta taattcaggt aaattggaagagtttgttca agggaacctt gagagagaat gtatggaagaaaagtgtagt tttgaagaag cacgagaagt ttttgaaaacactgaaagaa caactgaatt ttggaagcag tatgttgatggagatcagtg tgagtccaat ccatgtttaa atggcggcagttgcaaggat gacattaatt cctatgaatg ttggtgtccctttggatttg aaggaaagaa ctgtgaatta gatgtaacatgtaacattaa gaatggcaga tgcgagcagt tttgtaaaaatagtgctgat aacaaggtgg tttgctcctg tactgagggatatcgacttg cagaaaacca gaagtcctgt gaaccagcagtgccatttcc atgtggaaga gtttctgttt cacaaacttctaagctcacc cgtgctgaga ctgtttttcc tgatgtggactatgtaaatt ctactgaagc tgaaaccatt ttggataacatcactcaaag cacccaatca tttaatgact tcactcgggttgttggtgga gaagatgcca aaccaggtca attcccttggcaggttgttt tgaatggtaa agttgatgca ttctgtggaggctctatcgt taatgaaaaa tggattgtaa ctgctgcccactgtgttgaa actggtgtta aaattacagt tgtcgcaggtgaacataata ttgaggagac agaacataca gagcaaaagcgaaatgtgat tcgaattatt cctcaccaca actacaatgcagctattaat aagtacaacc atgacattgc ccttctggaactggacgaac ccttagtgct aaacagctac gttacacctatttgcattgc tgacaaggaa tacacgaaca tcttcctcaaatttggatct ggctatgtaa gtggctgggg aagagtcttccacaaaggga gatcagcttt agttcttcag taccttagagttccacttgt tgaccgagcc acatgtcttc gatctacaaagttcaccatc tataacaaca tgttctgtgc tggcttccatgaaggaggta gagattcatg tcaaggagat agtgggggaccccatgttac tgaagtggaa gggaccagtt tcttaactggaattattagc tggggtgaag agtgtgcaat gaaaggcaaatatggaatat ataccaaggt atcccggtat gtcaactgga ttaaggaaaa aacaaagctc act

As used herein, polypeptide refers to a polymer of amino acids and doesnot refer to a specific length of the polymer; thus, peptides,oligopeptides, and proteins are included within the definition ofpolypeptide. In one embodiment a polypeptide is a single-chainpolypeptide. As used herein, a protein can be a single-chain polypeptideor it can include more than one single-chain polypeptide. Also as usedherein, the term polypeptide can include polypeptides that have one ormore post-expression modifications, for example, glycosylation,acetylation, phosphorylation, pegylation, addition of a lipid moiety, orthe addition of any organic or inorganic molecule. Amino acidsspecifically include but are not limited to common so-called naturallyoccurring amino acids. These include alanine, arginine, asparagine,aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine. Included within thedefinition of polypeptide are, for example, polypeptides containing oneor more analogs of an amino acid (including, for example, unnaturalamino acids) and polypeptides with substituted linkages, as well asother modifications known in the art, both naturally occurring andnon-naturally occurring. In one embodiment a polypeptide is a fusionprotein.

Various changes may be made in the amino acid sequences of thepolypeptides and proteins or components thereof of the invention, orcorresponding DNA sequences encoding such polypeptides and proteins,without appreciable loss of their biological activity, function, orutility. Derivatives, analogs, or mutants resulting from such changesand the use of such derivatives are within the scope of the presentinvention.

As will be appreciated by those of skill in the art, substitutes for anamino acid may be selected from other members of a class to which theamino acid belongs (see, e.g., Table 1). Furthermore, various aminoacids are commonly substituted with neutral amino acids, e.g., alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine. See, e.g., MacLennan D H et al., Acta Physiol Scand Suppl.643:55-67 (1988); Sasaki N et al., Adv Biophys. 35:1-24 (1998).

TABLE 1 Original Exemplary Typical Residue Substitutions SubstitutionAla (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln Gln Asp(D) Glu Glu Cys (C) Ser, Ala Ser Gln (Q) Asn Asn Gly (G) Pro, Ala AlaHis (H) Asn, Gln, Lys, Arg Lys Ile (I) Leu, Val, Met, Ala, Phe,Norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K)Arg, 1,4-Diamino-butyric Acid, Gln, Asn Arg Met (M) Leu, Phe, Ile LeuPhe (F) Leu, Val, Ile, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr, Ala,Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, SerPhe Val (V) Ile, Met, Leu, Phe, Ala, Norleucine Leu

A fusion protein, as used herein, refers to a recombinant fusion proteinencompassing at least two heterologous polypeptides. A recombinantfusion protein refers to a protein encoded by a single nucleotidesequence derived from at least two heterologous nucleotide sequencescovalently linked to one another such that coding sequence from eachcomponent nucleotide sequence is translated in its proper reading frame.General methods for making recombinant DNA constructs are well known inthe art. As disclosed below, a fusion protein is a type of a conjugateas used herein. Fusion proteins in one embodiment can optionally bemodified to include one or more carbohydrate or other non-proteinaceousmoieties.

As described in greater detail below, a fusion protein can optionallyinclude a linker moiety between any of the component amino acidsequences. The optional linker moiety can but need not include one ormore amino acids. In one embodiment the optional linker moiety is apeptide at least two amino acid residues long.

Fusion proteins of the invention generally include proFIX fusionproteins. In one embodiment the proFIX fusion protein is aproFIX-albumin fusion protein. A proFIX-albumin fusion protein as usedherein refers to a fusion protein that includes a proFIX polypeptidecovalently bonded to albumin. The proFIX polypeptide can be fused toeither the N-terminal end of an albumin polypeptide or to the C-terminalend of an albumin polypeptide, provided the proFIX component of theproFIX-albumin fusion protein can be processed by enzymatically activePC5 to yield a mature FIX-containing polypeptide, as described herein.In one embodiment the proFIX polypeptide is a human proFIX polypeptideand the albumin polypeptide is a human albumin polypeptide.

In another embodiment the proFIX fusion protein is a proFIX-transferrinfusion protein. A proFIX-transferrin fusion protein as used hereinrefers to a fusion protein that includes a proFIX polypeptide covalentlybonded to transferrin. The proFIX polypeptide can be fused to either theN-terminal end of an transferrin polypeptide or to the C-terminal end ofan transferrin polypeptide, provided the proFIX component of theproFIX-transferrin fusion protein can be processed by enzymaticallyactive PC5 to yield a mature FIX-containing polypeptide, as describedherein. In one embodiment the proFIX polypeptide is a human proFIXpolypeptide and the transferrin polypeptide is a human transferrinpolypeptide.

Featured proFIX fusion proteins of the invention include proFIX fusionproteins that can be specifically bound by a neonatal Fc receptor(FcRn). These featured proFIX fusion proteins include a proFIX-FcRnbinding partner fusion protein, a proFIX-Fc fusion protein, a proFIX-Fchomodimer, and a proFIX-Fc monomer-dimer hybrid (see, e.g., FIG. 2).

As used herein, FcRn or, equivalently, FcRn receptor, refers to neonatalFc receptor. FcRn was first described as an enterocyte receptor for IgGin neonatal rats and mice. This receptor binds to the Fc portion ofimmunoglobulin G (IgG) and transports IgG by transcytosis in a luminalto serosal direction, i.e., from gut lumen to interstitium, in a processbelieved to be responsible for delivery of maternal IgG to the neonate.FcRn-mediated transport of IgG is believed to underlie passiveacquisition of IgG during the newborn suckling period. It wassubsequently discovered that FcRn is widely expressed on multiple typesof human epithelia, not only in newborns but also in adults.

The FcRn has been the basis for systemic delivery of a number ofmolecules provided as conjugates with FcRn binding partners. See U.S.Pat. Nos. 6,030,613, 6,086,875, and 6,485,726, and publishedinternational patent application WO 03/077834. The FcRn now is wellcharacterized. The FcRn has been isolated for several mammalian species,including humans. Sequences of the human, rat, and mouse FcRn moleculescan be found, for example, in Story C M et al. (1994) J Exp Med.180:2377-81. The FcRn binds IgG (but not other immunoglobulin classessuch as IgA, IgD, IgM and IgE) at a relatively lower pH, activelytransports the IgG transcellularly in a luminal to serosal direction,and then releases the IgG at a relatively higher pH found in theinterstitial fluids. As will be recognized by those of ordinary skill inthe art, FcRn can be isolated by cloning or by affinity purificationusing, for example, monoclonal antibodies. Such isolated FcRn then canbe used to identify and isolate FcRn binding partners.

As used herein, an FcRn binding partner means any entity that can bespecifically bound by an FcRn. The FcRn binding partner in certainembodiments can include but is not limited to whole IgG, an Fc fragmentof IgG, and other fragments of IgG that include the complete bindingregion for the FcRn. The region of the Fc portion of IgG that binds tothe FcRn has been described based upon X-ray crystallography (BurmeisterW P et al., Nature 372:379-83 (1994)). The major contact area of Fc withthe FcRn is near the junction of the C_(H)2 and C_(H)3 domains.Following the numbering convention for amino acids based on Kabat at al.(Sequences of Proteins of Immunological Interest, U.S. Department ofPublic Health, Bethesda, Md., 1991), potential contacts are residues248, 250-257, 272, 285, 288, 290-291, 308-311 and 314 in C_(H)2 and385-387, 428 and 433-436 in C_(H)3. These sites are distinct from thoseidentified by subclass comparison or by site-directed mutagenesis asimportant for Fc binding to leukocyte FcγRI and FcγRII. The foregoingFc-FcRn contacts are all within a single Ig heavy chain. It has beennoted previously that two FcRn can bind a single (homodimeric) Fcmolecule. The crystallographic data suggest that in such a complex, eachFcRn molecule binds a single polypeptide of the Fc homodimer FcRnbinding partner. Thus in one embodiment a fragment of IgG that includesthe complete binding region for the FcRn includes at least the majorcontact area of Fc with the FcRn near the junction of the C_(H)2 andC_(H)3 domains.

In one embodiment an FcRn binding partner is an FcRn binding partnerother than a whole IgG. In one embodiment an FcRn binding partner is anFcRn binding partner other than a whole human IgG.

In one embodiment an FcRn binding partner is an Fc fragment of IgG.Except as specified otherwise herein, an Fc fragment of IgG is ahomodimer of IgG heavy chain polypeptide fragments (i.e., Fcpolypeptides) which, when associated, together constitute a fragment ofwhole IgG that includes the hinge, C_(H)2, and C_(H)3 domains of IgG. AnFc fragment of IgG corresponds to a proteolytic fragment of IgGcontaining only the disulfide-linked carboxyl terminal regions of thetwo heavy chains of IgG. Fc fragments of IgG mediate effector functionsby binding to certain Fc receptors and C1q complement protein, and theyare to be distinguished from antigen-binding Fab fragments of IgG. Inone embodiment an FcRn binding partner is an Fc fragment of a human IgG,i.e., a human Fc gamma. In a particular embodiment an FcRn bindingpartner is an Fc fragment of a human IgG1 (i.e., Fcγ1). In oneembodiment each polypeptide of an Fc fragment (i.e., each Fcpolypeptide) of a human IgG has an amino acid sequence as provided bySEQ ID NO:5, corresponding to Kabat amino acid residue numbers 221-447.

SECS ID NO: 5DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD 60GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK 120GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS 180DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK 227

In one embodiment a nucleic acid encoding a Fc polypeptide from humanIgG1 has a sequence provided by GenBank accession no. Y14735 (SEQ IDNO:62):

SEQ ID NO: 62 gacaaaactc acacatgccc accgtgccca gcacctgaactcctgggggg accgtcagtc ttcctettcc ccccaaaacccaaggacacc ctcatgatct cccggacccc tgaggtcacatgcgtggtgg tggacgtgag ccacgaagac cctgaggtcaagttcaactg gtacgtggac ggcgtggagg tgcataatgccaagacaaag ccgcgggagg agcagtacaa cagcacgtaccgtgtggtca gcgtcctcac cgtcctgcac caggactggctgaatggcaa ggagtacaag tgcaaggtct ccaacaaagccctcccagcc cccatcgaga aaaccatctc caaagccaaagggcagcccc gagaaccaca ggtgtacacc ctgcccccatcccgggatga gctgaccaag aaccaggtca gcctgacctgcctggtcaaa ggcttctatc ccagcgacat cgccgtggagtgggagagca atgggcagcc ggagaacaac tacaagaccacgcctcccgt gctggactcc gacggctcct tcttcctctacagcaagctc accgtggaca agagcaggtg gcagcaggggaacgtcttct catgctccgt gatgcatgag gctctgcacaaccactacac gcagaagagc ctctccctgt ctccgggtaa a

The nucleotide sequence of the Fc polypeptide can be modified toincorporate or eliminate restriction endonuclease sites while preservingthe amino acid sequence. In one embodiment, the codons for A231, P232,and E233 are modified from GCA, CCT, GAA to GCT, CCG, GAA in order toincorporate a BspEI restriction site without altering the expressedamino acid sequence. In another embodiment, the codons for G236, G237,and P238 are modified from GGG, GGA, CCG to GGC, GGA, CCG in order toincorporate a RsrII restriction site while preserving the amino acidsequence.

An Fc polypeptide of IgG can be modified according to well recognizedprocedures such as site-directed mutagenesis and the like to yieldmodified IgG or Fc fragments or portions thereof that will be bound byFcRn. Such modifications include modifications remote from the FcRncontact sites as well as modifications within the contact sites thatpreserve or even enhance binding to the FcRn. For example, the followingsingle amino acid residues (Kabat numbering convention) in human IgG1 Fc(Fcγ1) can be substituted without significant loss of Fc bindingaffinity for FcRn: P238A, S239A, K246A, K248A, D249A, M252A, T256A,E258A, T260A, D265A, S267A, H268A, E269A, D270A, E272A, L274A, N276A,Y278A, D280A, V282A, E283A, H285A, N286A, T289A, K290A, R292A, E293A,E294A, Q295A, Y296F, N297A, S298A, Y300F, R301A, V303A, V305A, T307A,L309A, Q311A, D312A, N315A, K317A, E318A, K320A, K322A, S324A, K326A,A327Q, P329A, A330Q, P331A, E333A, K334A, T335A, S337A, K338A, K340A,Q342A, R344A, E345A, Q347A, R355A, E356A, M358A, T359A, K360A, N361A,Q362A, Y373A, S375A, D376A, A378Q, E380A, E382A, S383A, N384A, Q386A,E388A, N389A, N390A, Y391F, K392A, L398A, S400A, D401A, D413A, K414A,R416A, Q418A, Q419A, N421A, V422A, S424A, E430A, N434A, T437A, Q438A,K439A, S440A, S444A, and K447A, where for example P238A representswild-type proline substituted by alanine at position number 238. As anexample, one specific embodiment, incorporates the N297A mutation,removing a highly conserved N-glycosylation site. In addition to alanineother amino acids may be substituted for the wild-type amino acids atthe positions specified above. Mutations may be introduced singly intoFc giving rise to more than one hundred FcRn binding partners distinctfrom native Fc. Additionally, combinations of two, three, or more ofthese individual mutations may be introduced together, giving rise tohundreds more FcRn binding partners. Moreover, one of thepolynucleotides of a dimeric Fc fragment or other dimeric FcRn bindingpartner may include a mutation while the other does not, or both may bemutated but with different mutations. Any of the mutations describedherein, including N297A, may be used to modify Fc.

Certain of the above mutations may confer new functionality upon theFcRn binding partner. For example, one embodiment incorporates N297A,removing a highly conserved N-glycosylation site. The potential effectof this mutation is to reduce immunogenicity, thereby enhancingcirculating half-life of the FcRn binding partner, and to render theFcRn binding partner incapable of binding to other Fc receptorsincluding FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA, without compromisingaffinity for FcRn. Routledge E G et al. (1995) Transplantation60:847-53; Friend P J et al. (1999) Transplantation 68:1632-7; Shields RL et al. (1995) J Biol Chem 276:6591-604. As a further example of newfunctionality arising from mutations described above, affinity for FcRnmay be increased beyond that of wild-type in some instances. Thisincreased affinity may reflect an increased “on” rate, a decreased “off”rate, or both an increased “on” rate and a decreased “off” rate.Mutations reported to impart an increased affinity for FcRn includeT256A, T307A, E380A, and N434A. Shields et al. (2001) J Biol Chem276:6591.

Additionally, at least three human Fc gamma receptors appear torecognize a binding site on IgG within the lower hinge region, generallyamino acids 234-237. Therefore, another example of new functionality andpotential decreased immunogenicity may arise from mutations of thisregion, as for example by replacing amino acids 233-236 of human IgG1(ELLG; SEQ ID NO:6) to the corresponding sequence from IgG2 (PVA, withone amino acid deletion). It has been reported that FcγRI, FcγRII, andFcγRIII, which mediate various effector functions, will not bind to IgG1when such mutations have been introduced. Ward E S et al. (1995) TherImmunol 2:77-94; Armour K L et al. (1999) Eur J Immunol 29:2613-24.

As used herein, a proFIX-FcRn binding partner fusion protein refers to afusion protein that includes a proFIX polypeptide covalently linked toan FcRn binding partner polypeptide. The proFIX polypeptide component iscapable of being processed by PC5 to yield a mature form of FIX that inturn is capable of being activated by Factor XIa to yield an activatedFIXa polypeptide. The FcRn binding partner polypeptide component can beany suitable polypeptide that can be specifically bound by FcRn. Incertain embodiments the FcRn binding partner polypeptide is an Fcpolypeptide of IgG or an Fc polypeptide of a human IgG. The linkagebetween the proFIX polypeptide component and the FcRn binding partnerpolypeptide component can be direct or it can be indirect via a linker.

As used herein, a proFIX-Fc fusion protein refers to a fusion proteinthat includes a proFIX polypeptide covalently linked to an Fcpolypeptide. The proFIX polypeptide component is capable of beingprocessed by PC5 to yield a mature form of FIX that in turn is capableof being activated by Factor XIa to yield an activated FIXa polypeptide.The Fc polypeptide component can be any suitable Fc polypeptide that canbe specifically bound by FcRn. In certain embodiments the Fc polypeptideis an Fc polypeptide of IgG or an Fc polypeptide of a human IgG. Thelinkage between the proFIX polypeptide component and the Fc polypeptidecomponent can be direct or it can be indirect via a linker.

Fusion proteins of the invention can include various dimeric structuresformed through dimerization of identical or nonidentical polypeptides.Thus dimeric fusion proteins of the invention can include homodimers andheterodimers. In one embodiment, the fusion protein of the inventioncomprises a first polypeptide chain comprising at least a first domain,said first domain having at least one specific binding partner, and asecond polypeptide chain comprising at least a second domain, whereinsaid second domain is a specific binding partner of said first domain.The fusion protein thus comprises a polypeptide capable of dimerizingwith another polypeptide due to the interaction of the first domain andthe second domain. Methods of dimerizing antibodies using heterologousdomains are known in the art. See, e.g., U.S. Pat. Nos. 5,807,706 and5,910,573; Kostelny et al. (1992) J Immunol 148:1547.

Dimerization can occur by formation of a covalent bond, or alternativelya non-covalent bond, e.g., hydrophobic interaction, Van der Waalsforces, interdigitation of amphiphilic peptides such as, but not limitedto, alpha helices, charge-charge interactions of amino acids bearingopposite charges, such as, but not limited to, lysine and aspartic acid,arginine and glutamic acid. In one embodiment, the dimerization involvesa helix bundle comprising a helix, a turn, and another helix. In anotherembodiment, the dimerization involves a leucine zipper comprising apeptide having several repeating amino acids in which every seventhamino acid is a leucine residue. In one embodiment, the specific bindingpartners are Fos and Jun. See Branden et al. (1991) Introduction toProtein Structure, Garland Publishing, New York.

In another embodiment, dimerization is mediated by a chemical linkage(see, e.g., Brennan et al. (1985) Science 229:81). In this embodiment,intact immunoglobulins, or fusion proteins comprised of at least aportion of an immunoglobulin constant region, are cleaved to generateheavy chain fragments. These fragments are reduced in the presence of adithiol complexing agent such as sodium arsenite to stabilize vicinaldithiols and prevent intermolecular disulfide formation. The fragmentsso generated are then converted to thionitrobenzoate (TNB) derivatives.One of the TNB derivatives is then reconverted to the heavy chainfragment thiol by reduction with a reducing agent such asmercaptoethylamine and is then mixed with an equimolar amount of theother TNB derivative to form a dimer.

As used herein, a proFIX-Fc homodimer refers to a covalently ornoncovalently linked dimer formed between two identical proFIX-Fcpolypeptides. In one embodiment the two polypeptides are covalentlylinked to one another so as to form a duplex structure in which the twopolypeptides are co-aligned from N-terminus to C-terminus. In oneembodiment the two polypeptides are covalently linked to one anotherthrough one or more disulfide bridges formed between the hinge domainregion of one Fc polypeptide and the hinge domain region of the other Fcpolypeptide, so as to form a duplex structure in which the Fcpolypeptides are co-aligned from N-terminus to C-terminus. A diagramillustrating the structure of one embodiment of a proFIX-Fc homodimer isprovided in FIG. 2A.

As used herein, a proFIX-Fc monomer-dimer hybrid refers to aheterodimeric protein that includes a first polypeptide chain and asecond polypeptide chain, wherein the first polypeptide chain includes aproFIX polypeptide and at least a portion of an immunoglobulin constantchain capable of binding specifically to an FcRn, and wherein the secondpolypeptide chain includes at least a portion of an immunoglobulinconstant chain capable of binding specifically to an FcRn but whichsecond polypeptide chain does not include a proFIX polypeptide. Thus inthis aspect of the invention the protein is monomeric with respect toproFIX and dimeric with respect to Fc (hence the term “monomer-dimerhybrid”). However, it should be appreciated that, while a proFIX-Fcmonomer-dimer hybrid is heterodimeric since the two chains are distinct,these molecules may also be referred to as “monomer” for short sincethere is only one biologically active proFIX compared to traditionalhomodimeric Fc fusion proteins. Reference is made to US 2005/0032174,the entire content of which is incorporated herein by reference. In oneembodiment the portion of an immunoglobulin constant chain capable ofbinding specifically to an FcRn includes, in each instance, an IgG Fcfragment domain. In one embodiment the portion of an immunoglobulinconstant chain capable of binding specifically to an FcRn is, in eachinstance, an IgG Fc fragment domain. In one embodiment the portion of animmunoglobulin constant chain capable of binding specifically to an FcRnincludes, in each instance, a human IgG Fc fragment domain. In oneembodiment the portion of an immunoglobulin constant chain capable ofbinding specifically to an FcRn is, in each instance, a human IgG Fcfragment domain. In one embodiment the portion of an immunoglobulinconstant chain capable of binding specifically to an FcRn in the firstpolypeptide chain is identical to the portion of an immunoglobulinconstant chain capable of binding specifically to an FcRn in the secondpolypeptide chain. In one embodiment the portion of an immunoglobulinconstant chain capable of binding specifically to an FcRn in the firstpolypeptide chain is not identical to the portion of an immunoglobulinconstant chain capable of binding specifically to an FcRn in the secondpolypeptide chain. A diagram illustrating the structure of oneembodiment of a proFIX-Fc monomer-dimer hybrid is provided in FIG. 2B.

The fusion proteins and conjugates of the invention can optionallycomprise at least one linker molecule. A linker generally can becomprised of any organic molecule. In one embodiment, the linker ispolyethylene glycol (PEG). In another embodiment, the linker iscomprised of amino acids. An amino acid linker can comprise 1-5 aminoacids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100amino acids, 100-200 amino acids. An amino acid linker in one embodimentcan be encoded by a nucleic acid sequence incorporated into a nucleicacid molecule encoding a fusion protein. An amino acid linker in oneembodiment can be generated as an isolated synthetic peptide, i.e., apeptide produced using known chemical synthesis techniques (e.g., solidphase synthesis) performed outside of a cell. Any of the amino acidlinkers described herein may be used in a fusion protein of theinvention, e.g., a proFIX-Fc monomer-dimer hybrid.

In one embodiment, an amino acid linker is the eight amino acid linkerEFAGAAAV (SEQ ID NO:7), wherein E represents glutamic acid, F representsphenylalanine, A represents alanine, G represents glycine, and Vrepresents valine. In one embodiment the linker is encoded by a nucleicacid sequence that is incorporated into a nucleic acid molecule encodinga fusion protein.

An amino acid linker in certain embodiments can include the sequenceG_(n) (SEQ ID NO:8), (GA)_(n) (SEQ ID NO:9), (GUS)_(n) (SEQ ID NO:10),(GGGGS)_(n) (SEQ ID NO:11), or any combination thereof, wherein in eachinstance G represent glycine, A represents alanine, S represents serine,and n may be an integer from 1-10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQID NO:12), GGSGGSGGSGGSGGG (SEQ ID NO:13), GGSGGSGGGGSGGGGS (SEQ IDNO:14), GGSGGSGGSGGSGGSGGS (SEQ ID NO:15). In one embodiment the linkeris GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:16). In one embodiment thelinker is encoded by a nucleic acid sequence that is incorporated into anucleic acid molecule encoding a fusion protein. The linker does noteliminate or diminish the biological activity of the fusion protein.Optionally, the linker enhances the biological activity of the fusionprotein, e.g., by further diminishing the effects of steric hindranceand making the FIX component and/or other component more accessible.

The linker may also incorporate a moiety capable of being cleaved eitherchemically (e.g., hydrolysis of an ester bond), enzymatically (e.g.,incorporation of a protease cleavage sequence) or photolytically (e.g.,a chromophore such as 3-amino-3-(2-nitrophenyl) proprionic acid (ANP))in order to release the biologically active molecule from the Fcprotein.

The chemistry of cross-linking and effective reagents for such purposesare well known in the art. The nature of the crosslinking reagent usedto conjugate various components is not restricted by the invention. Anycrosslinking agent may be used provided that a) the activity of the FIXis retained, and b) binding by the FcRn of the FcRn binding partnercomponent of the conjugate is not adversely affected.

An example of an effective one-step crosslinking of Fc and a compound isoxidation of Fc with sodium periodate in sodium phosphate buffer for 30minutes at room temperature, followed by overnight incubation at 4° C.with the compound to be conjugated. Conjugation also may be performed byderivatizing both the compound and Fc fragments with sulfosuccinimidyl6-[3-(2-pyridyldithio)propionamide]hexanoate (sulfo-LC-SPDP, Pierce) for18 hours at room temperature. Conjugates also may be prepared byderivatizing Fc fragments and the desired compound to be delivered withdifferent crosslinking reagents that will subsequently form a covalentlinkage. An example of this reaction is derivatization of Fc fragmentswith sulfosuccinimidyl 4-(N-maleimidomethyl)cyclo-hexane-1-carboxylate(Sulfo-SMCC, Pierce) and the compound to be conjugated to Fc isthiolated with N-succinimidyl S-acetylthioacetate (SATA). Thederivatized components are purified free of crosslinker and combined atroom temperature for one hour to allow crosslinking. Other crosslinkingreagents comprising aldehyde, imide, cyano, halogen, carboxyl, activatedcarboxyl, anhydride, and maleimide functional groups are known topersons of ordinary skill in the art and also may be used forconjugation of compounds to Fc fragments.

The choice of cross-linking reagent will, of course, depend on thenature of the components desired to be conjugated. The crosslinkingreagents described above are effective for protein-protein conjugations.If a carbohydrate or carbohydrate-containing moiety is to be conjugatedto a polypeptide, then heterobifunctional crosslinking reagents such asABH, M2C2H, MPBH and PDPH are useful for conjugation (Pierce ChemicalCo., Rockford, Ill.). Another method of conjugating proteins andcarbohydrates is disclosed by Brumeanu et al. (Genetic Engineering News,Oct. 1, 1995, p. 16). If a lipid or a lipid-containing moiety is to beconjugated to a polypeptide, then crosslinkers such as SPDP, SMPB, andderivatives thereof may be used (Pierce Chemical Co., Rockford, Ill.).

In all of the above crosslinking reactions it is important to purify thederivatized compounds free of crosslinking reagent. It is important alsoto purify the final conjugate substantially free of unconjugatedreactants. Purification may be achieved by affinity, gel filtration orion exchange chromatography based on the properties of either componentof the conjugate. A particularly preferred method is an initial affinitypurification step using protein A-Sepharose to retain Fc andFc-containing conjugates, followed by gel filtration or ion exchangechromatography based on the mass, size or charge of the Fc conjugate.The initial step of this purification scheme ensures that the conjugatewill bind to FcRn which is an essential requirement of the invention.

It has been discovered according to the invention that PC5 can be usedto process a FIX precursor polypeptide to yield a mature FIXpolypeptide. In one embodiment both the PC5 and the FIX precursorpolypeptide are expressed within a cell, where the PC5 processes the FIXprecursor polypeptide. In another embodiment an isolated PC5 can be usedto process an isolated FIX precursor polypeptide.

When the PC5 and the FIX precursor polypeptide are to be coexpressedwithin a cell, in one embodiment the cell naturally expresses PC5 andcontains an expression vector encoding a proprotein of Factor IX(proFIX), or a fusion protein thereof.

When the PC5 and the FIX precursor polypeptide are to be coexpressedwithin a cell, in one embodiment the cell contains an expression vectorencoding a proprotein of Factor IX (proFIX), or a fusion proteinthereof, and the cell is modified by homologous recombination to includea nucleic acid expression control sequence that causes overexpression ofan endogenous PC5 gene. In this embodiment an exogenous, more potentpromoter is exchanged for the endogenous PC5 promoter, resulting inoverexpression of endogenous PC5. See, e.g., U.S. Pat. Nos. 5,641,670,5,733,761, and 5,272,071; WO 91/06666; WO 91/06667; and WO 90/11354, allof which are incorporated by reference in their entirety.

When the PC5 and the FIX precursor polypeptide are to be coexpressedwithin a cell, in one embodiment the cell contains an expression vectorencoding a proprotein of Factor IX (proFIX), or a fusion proteinthereof, and an expression vector encoding a functional PC5 polypeptide.The expression vector encoding a proprotein of Factor IX (proFIX), or afusion protein thereof, and the expression vector encoding a functionalPC5 polypeptide can be separate expression vectors or can be combined ina single expression vector.

As used herein, an expression vector refers to any nucleic acidconstruct which contains the necessary elements for the transcriptionand translation of an inserted coding sequence, or in the case of an RNAviral vector, the necessary elements for replication and translation,when introduced into an appropriate host cell. Expression vectors caninclude plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of the invention will include a polynucleotideencoding a polypeptide of the invention, e.g., PC5 and proFIX. As usedherein, a polynucleotide refers to any covalently linked polymer of twoor more nucleotides. A nucleotide is a molecule composed of a sugar(e.g., ribose or deoxyribose) linked to a phosphate group and anucleobase, i.e., either a purine or a pyrimidine. Purines include butare not limited to adenine and guanine. Pyrimidines include but are notlimited to cytosine, thymine, and uracil. In one embodiment apolynucleotide is a polymer of deoxyribonucleotides, i.e., a DNAmolecule. In one embodiment a polynucleotide is a polymer ofribonucleotides, i.e., an RNA molecule. A polynucleotide in oneembodiment is a nucleic acid molecule that encodes a polypeptide, i.e.,the polynucleotide is a coding sequence. In one embodiment a nucleicacid molecule that encodes a polypeptide is a genomic DNA molecule thatencodes the polypeptide. In one embodiment a nucleic acid molecule thatencodes a polypeptide is a complementary DNA (cDNA) molecule thatencodes the polypeptide. In one embodiment a polynucleotide is arecombinant DNA molecule that encodes a polypeptide.

In certain embodiments a coding sequence is operably linked to anexpression control sequence. As used herein, two nucleic acid sequencesare operably linked when they are covalently linked in such a way as topermit each component nucleic acid sequence to retain its functionality.A coding sequence and a gene expression control sequence are said to beoperably linked when they are covalently linked in such a way as toplace the expression or transcription and/or translation of the codingsequence under the influence or control of the gene expression controlsequence. Two DNA sequences are said to be operably linked if inductionof a promoter in the 5′ gene expression sequence results in thetranscription of the coding sequence and if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion to direct the transcription of the coding sequence, or (3)interfere with the ability of the corresponding RNA transcript to betranslated into a protein. Thus, a gene expression sequence would beoperably linked to a coding nucleic acid sequence if the gene expressionsequence were capable of effecting transcription of that coding nucleicacid sequence such that the resulting transcript is translated into thedesired protein or polypeptide.

A gene expression control sequence as used herein is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the coding nucleic acid to which it is operably linked.The gene expression control sequence may, for example, be a mammalian orviral promoter, such as a constitutive or inducible promoter.Constitutive mammalian promoters include, but are not limited to, thepromoters for the following genes: hypoxanthine phosphoribosyltransferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actinpromoter, and other constitutive promoters. Exemplary viral promoterswhich function constitutively in eukaryotic cells include, for example,promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40),papilloma virus, adenovirus, human immunodeficiency virus (HIV), Roussarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus, and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression control sequence shall include, asnecessary, 5′ non-transcribing and 5′ non-translating sequences involvedwith the initiation of transcription and translation, respectively, suchas a TATA box, capping sequence, CAAT sequence, and the like.Especially, such 5′ non-transcribing sequences will include a promoterregion which includes a promoter sequence for transcriptional control ofthe operably joined coding nucleic acid. The gene expression sequencesoptionally include enhancer sequences or upstream activator sequences asdesired.

Viral vectors include, but are not limited to, nucleic acid sequencesfrom the following viruses: retrovirus, such as Moloney murine leukemiavirus, Harvey murine sarcoma virus, murine mammary tumor virus, and Roussarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses;polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus;vaccinia virus; polio virus; and RNA virus such as a retrovirus. One canreadily employ other vectors not named but known in the art. Certainviral vectors are based on non-cytopathic eukaryotic viruses in whichnon-essential genes have been replaced with the gene of interest.Non-cytopathic viruses include retroviruses, the life cycle of whichinvolves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H.Freeman Co., New York (1990) and Murry, E. J., Methods in MolecularBiology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment the virus is an adeno-associated virus, adouble-stranded DNA virus. The adeno-associated virus can be engineeredto be replication-deficient and is capable of infecting a wide range ofcell types and species. It further has advantages such as heat and lipidsolvent stability; high transduction frequencies in cells of diverselineages, including hemopoietic cells; and lack of superinfectioninhibition thus allowing multiple series of transductions. Reportedly,the adeno-associated virus can integrate into human cellular DNA in asite-specific manner, thereby minimizing the possibility of insertionalmutagenesis and variability of inserted gene expression characteristicof retroviral infection. In addition, wild-type adeno-associated virusinfections have been followed in tissue culture for greater than 100passages in the absence of selective pressure, implying that theadeno-associated virus genomic integration is a relatively stable event.The adeno-associated virus can also function in an extrachromosomalfashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operably encoded within the plasmid. Somecommonly used plasmids available from commercial suppliers includepBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMVplasmids, pSV40, and pBlueScript. Additional examples of specificplasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro,catalog number V87020; pcDNA4/myc-His, catalog number V86320; andpBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad,Calif.). Other plasmids are well-known to those of ordinary skill in theart. Additionally, plasmids may be custom designed using standardmolecular biology techniques to remove and/or add specific fragments ofDNA.

In one insect expression system that may be used to produce the proteinsof the invention, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express the foreign genes. The virusgrows in Spodoptera frugiperda cells. A coding sequence may be clonedinto non-essential regions (for example, the polyhedron gene) of thevirus and placed under control of an ACNPV promoter (for example, thepolyhedron promoter). Successful insertion of a coding sequence willresult in inactivation of the polyhedron gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedron gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (see, e.g., Smith et al. (1983) J Virol 46:584; U.S.Pat. No. 4,215,051). Further examples of this expression system may befound in Ausubel et al., eds. (1989) Current Protocols in MolecularBiology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

Another system which can be used to express the proteins of theinvention is the glutamine synthetase gene expression system, alsoreferred to as the “GS expression system” (Lonza Biologics PLC,Berkshire UK). This expression system is described in detail in U.S.Pat. No. 5,981,216.

In mammalian host cells, a number of viral based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, a coding sequence may be ligated to an adenovirustranscription/translation control complex, e.g., the late promoter andtripartite leader sequence. This chimeric gene may then be inserted inthe adenovirus genome by in vitro or in vivo recombination. Insertion ina non-essential region of the viral genome (e.g., region E1 or E3) willresult in a recombinant virus that is viable and capable of expressingpeptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc NatlAcad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may beused. See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415;Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc NatlAcad Sci USA 79:4927.

To increase efficiency of production, the polynucleotides can bedesigned to encode multiple units of the protein of the inventionseparated by enzymatic cleavage sites. The resulting polypeptide can becleaved (e.g., by treatment with the appropriate enzyme) in order torecover the polypeptide units. This can increase the yield ofpolypeptides driven by a single promoter. When used in appropriate viralexpression systems, the translation of each polypeptide encoded by themRNA is directed internally in the transcript; e.g., by an internalribosome entry site, IRES. Thus, the polycistronic construct directs thetranscription of a single, large polycistronic mRNA which, in turn,directs the translation of multiple, individual polypeptides. Thisapproach eliminates the production and enzymatic processing ofpolyproteins and may significantly increase yield of polypeptide drivenby a single promoter.

Vectors used in transformation will usually contain a selectable markerused to identify transformants. In bacterial systems, this can includean antibiotic resistance gene such as ampicillin or kanamycin.Selectable markers for use in cultured mammalian cells include genesthat confer resistance to drugs, such as neomycin, hygromycin, andmethotrexate. The selectable marker may be an amplifiable selectablemarker. One amplifiable selectable marker is the dihydrofolate reductase(DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA80:2495-9. Selectable markers are reviewed by Thilly (1986) MammalianCell Technology, Butterworth Publishers, Stoneham, Mass., and the choiceof selectable markers is well within the level of ordinary skill in theart.

Selectable markers may be introduced into the cell on a separate plasmidat the same time as the gene of interest, or they may be introduced onthe same plasmid. If on the same plasmid, the selectable marker and thegene of interest may be under the control of different promoters or thesame promoter, the latter arrangement producing a dicistronic message.Constructs of this type are known in the art (for example, U.S. Pat. No.4,713,339).

The expression vectors can encode for tags that permit for easypurification of the recombinantly produced protein. Examples include,but are not limited to vector pUR278 (Ruther et al. (1983) EMBO J.2:1791) in which coding sequences for the protein to be expressed may beligated into the vector in frame with the lac z coding region so that atagged fusion protein is produced; pGEX vectors may be used to expressproteins of the invention with a glutathione S-transferase (GST) tag.These proteins are usually soluble and can easily be purified from cellsby adsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The vectors include cleavage sites(thrombin or Factor Xa protease or PreScission Protease™ (Pharmacia,Peapack, N.J.)) for easy removal of the tag after purification.

The expression vector or vectors are then transfected or co-transfectedinto a suitable target cell, which will express the polypeptides.Transfection techniques known in the art include, but are not limitedto, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725),electroporation (Neumann et al. (1982) EMBO J. 1:841), andliposome-based reagents. A variety of host-expression vector systems maybe utilized to express the proteins described herein including bothprokaryotic or eukaryotic cells. These include, but are not limited to,microorganisms such as bacteria (e.g., E. coli) transformed withrecombinant bacteriophage DNA or plasmid DNA expression vectorscontaining an appropriate coding sequence; yeast or filamentous fungitransformed with recombinant yeast or fungi expression vectorscontaining an appropriate coding sequence; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containingan appropriate coding sequence; plant cell systems infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus ortobacco mosaic virus) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasmid) containing an appropriate coding sequence; oranimal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos,HeLa, and BHK cells).

In one embodiment the host cell is a eukaryotic cell. As used herein, aeukaryotic cell refers to any animal or plant cell having a definitivenucleus. Eukaryotic cells of animals include cells of vertebrates, e.g.,mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells ofplants specifically can include, without limitation, yeast cells. Aeukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

In certain embodiments the eukaryotic cell is a mammalian cell. Amammalian cell is any cell derived from a mammal. Mammalian cellsspecifically include but are not limited to mammalian cell lines. In oneembodiment the mammalian cell is a human cell. In one embodiment themammalian cell is a HEK 293 cell, which is a human embryonic kidney cellline. HEK 293 cells are available as CRL-1533 from American Type CultureCollection, Manassas, Va., and as 293-H cells, Catalog No. 11631-017 or293-F cells, Catalog No. 11625-019 from Invitrogen (Carlsbad, Calif.).In one embodiment the mammalian cell is a PER.C6® cell, which is a humancell line derived from retina. PER.C6® cells are available from Crucell(Leiden, The Netherlands). In one embodiment the mammalian cell is aChinese hamster ovary (CHO) cell. CHO cells are available from AmericanType Culture Collection, Manassas, Va. (e.g., CHO-K1; CCL-61). In oneembodiment the mammalian cell is a baby hamster kidney (BHK) cell. BHKcells are available from American Type Culture Collection, Manassas, Va.(e.g., CRL-1632).

In one embodiment a plasmid including a proFIX-Fc fusion coding sequenceand a selectable marker, e.g., zeocin resistance, is transfected intoHEK 293 cells, for production of FIX-Fc homodimer.

In one embodiment a first plasmid including a proFIX-Fc fusion codingsequence and a first selectable marker, e.g., a zeocin resistance gene,and a second plasmid including an Fc coding sequence and a secondselectable marker, e.g., a neomycin resistance gene, are cotransfectedinto HEK 293 cells, for production of FIX-Fc monomer-dimer hybrid. Thefirst and second plasmids can be introduced in equal amounts (i.e., 1:1ratio), or they can be introduced in unequal amounts.

In one embodiment a first plasmid including a proFIX-Fc fusion codingsequence and a first selectable marker, e.g., a zeocin resistance gene,and a second plasmid including an Fc coding sequence and a secondselectable marker, e.g., a neomycin resistance gene, and a third plasmidincluding a PC5 coding sequence and a third selectable marker, e.g., ahygromycin resistance gene, are cotransfected into HEK 293 cells, forproduction of FIX-Fc monomer-dimer hybrid. The first and second plasmidscan be introduced in equal amounts (i.e., 1:1 molar ratio), or they canbe introduced in unequal amounts.

In one embodiment a first plasmid, including a proFIX-Fc fusion codingsequence, an Fc coding sequence, and a first selectable marker, e.g., azeocin resistance gene, and a second plasmid including a PC5 codingsequence and a second selectable marker, e.g., a hygromycin resistancegene, are cotransfected into HEK 293 cells, for production of FIX-Fcmonomer-dimer hybrid. The promoters for the proFIX-Fc fusion codingsequence and the Fc coding sequence can be different or they can be thesame.

In one embodiment a first plasmid, including a proFIX-Fc fusion codingsequence, an Fc coding sequence, and a first selectable marker, e.g., azeocin resistance gene, and a second plasmid including an Fc codingsequence and a second selectable marker, e.g., a neomycin resistancegene, and a third plasmid including a PC5 coding sequence and a thirdselectable marker, e.g., a hygromycin resistance gene, are cotransfectedinto HEK 293 cells, for production of FIX-Fc monomer-dimer hybrid. Thepromoters for the proFIX-Fc fusion coding sequence and the Fc codingsequence in the first plasmid can be different or they can be the same.The first and second plasmids can be introduced in equal amounts (i.e.,1:1 molar ratio), or they can be introduced in unequal amounts.

In one embodiment, a truncated form of the FIX intron I (GenBankaccession no. NC_(—)000023) is included within the coding sequence ofFIX. This has previously been shown to increase the level of FIXexpression due to functional splicing sequences to present in theprecursor mRNA. Kurachi S et al. (1995) J Biol Chem. 270:5276-81.

In one embodiment transfected cells are stably transfected. These cellscan be selected and maintained as a stable cell line, using conventionaltechniques known to those of skill in the art.

Host cells containing DNA constructs of the protein are grown in anappropriate growth medium. As used herein, the term “appropriate growthmedium” means a medium containing nutrients required for the growth ofcells. Nutrients required for cell growth may include a carbon source, anitrogen source, essential amino acids, vitamins, minerals, and growthfactors. Optionally the media can contain one or more selection factors.Optionally the media can contain bovine calf serum or fetal calf serum(FCS). In one embodiment, the media contains substantially no IgG. Thegrowth medium will generally select for cells containing the DNAconstruct by, for example, drug selection or deficiency in an essentialnutrient which is complemented by the selectable marker on the DNAconstruct or co-transfected with the DNA construct. Cultured mammaliancells are generally grown in commercially available serum-containing orserum-free media (e.g., MEM, DMEM, DMEM/F12). In one embodiment themedium is CD293 (Invitrogen, Carlsbad, Calif.). In one embodiment, themedium is CD17 (Invitrogen, Carlsbad, Calif.). Selection of a mediumappropriate for the particular cell line used is within the level ofordinary skill in the art.

In one aspect the invention provides a method for producing a matureFactor IX-containing polypeptide from a proprotein of Factor IX, or afusion protein thereof.

As used herein, a mature Factor IX-containing polypeptide refers to apolypeptide that includes a mature form of Factor IX. In one embodimenta mature Factor IX-containing polypeptide refers to a polypeptide thatis a mature form of Factor IX. A mature form of Factor IX includes atleast a γ-carboxylated Gla domain and a catalytic domain, and excludesboth a prepeptide and a propeptide, as described herein. The mature formof Factor IX can but need not necessarily be an activated form of FactorIX. For example, in one embodiment a mature form of Factor IX isrepresented by amino acid residues 47-461 of SEQ ID NO:3. In oneembodiment at least ten of the twelve glutamic acid residues in the Gladomain are gamma carboxylated. When only ten of the twelve glutamic acidresidues in the Gla domain are gamma carboxylated, in one embodiment theten carboxylated glutamic residues are the first ten glutamic acidresidues in the Gla domain (e.g., Glu53, Glu54, Glu61, Glu63, Glu66,Glu67, Glu72, Glu73, Glu76, and Glu79 in SEQ ID NO:3). In one embodimentat least eleven of the twelve glutamic acid residues in the Gla domainare gamma carboxylated. In one embodiment all of the glutamic acidresidues in the Gla domain are gamma carboxylated.

In one embodiment according to this aspect of the invention, a matureFactor IX-containing polypeptide refers to a polypeptide that is amature form of Factor IX.

In one embodiment according to this aspect of the invention, the matureFactor IX-containing polypeptide is a FIX-Fc monomer-dimer hybrid, suchas is disclosed in co-owned U.S. patent application Ser. No. 10/841,250,published as US 2005/0032174, the entire content of which isincorporated herein by reference. In this embodiment the proproproteinfusion protein corresponds to a proFIX-Fc monomer-dimer hybrid.

Also provided is a mature FIX-Fc monomer-dimer hybrid produced accordingto the method of this aspect of the invention.

The method for producing mature Factor IX-containing polypeptide entailsculturing cells containing a first expression vector encoding aproprotein of Factor IX, or a fusion protein thereof, and a secondexpression vector encoding a functional PC5 polypeptide, as describedabove. The cells are cultured under conditions that allow expression ofboth the proFIX or the fusion protein thereof and the functional PC5polypeptide.

As used herein, culturing refers to maintaining living cells in vitrofor at least a definite time. Maintaining can but need not include anincrease in population of living cells. For example, cells maintained inculture can be static in population but still viable and capable ofproducing a desired product, e.g., a recombinant protein or recombinantfusion protein. Suitable conditions for culturing eukaryotic cells arewell known in the art and include appropriate selection of culturemedia, media supplements, temperature, pH, oxygen saturation, and thelike. For commercial purposes culturing can include the use of any ofvarious types of scale-up systems including shaker flasks, rollerbottles, hollow fiber bioreactors, stirred-tank bioreactors, airliftbioreactors, Wave bioreactors, and others.

The cell culture conditions are also selected to allow processing of theproFIX or fusion protein thereof by the functional PC5 polypeptide.Conditions that allow processing of the proFIX or fusion protein thereofby the functional PC5 polypeptide specifically may include the presenceof a source of vitamin K. For example, in one embodiment stablytransfected HEK 293 cells are cultured in CD293 media (Invitrogen,Carlsbad, Calif.) supplemented with 4 mM glutamine and 10 μg/L vitaminK₃.

As used herein, processing by functional PC5 refers to cleavage ofpropeptide from proFIX. In other words, processing by functional PC5refers to the PC5-mediated conversion of a proFIX-containing polypeptideto a mature Factor IX-containing polypeptide.

The invention in one aspect relates to a method for increasing yield ofa mature Factor IX-containing polypeptide from a proprotein of FactorIX, or a fusion protein thereof. As used herein with respect to matureFactor IX-containing polypeptide, increasing yield refers to inducing ameasurable increase in amount or activity of a mature FactorIX-containing polypeptide, obtained with PC5 and under specifiedconditions, as compared to a reference amount or activity of the matureFactor IX-containing polypeptide, obtained without PC5 and under thespecified conditions. In one embodiment the measurable increase is atleast 5 percent. In one embodiment the measurable increase is at least10 percent. In additional individual embodiments the measurable increaseis at least 20 percent, at least 30 percent, at least 40 percent, atleast 50 percent, at least 60 percent, at least 70 percent, at least 80percent, at least 90 percent, or at least 100 percent.

The invention in one aspect relates to a method for producing a matureFactor IX-containing polypeptide from a proprotein of Factor IX, or afusion protein thereof. As used herein, producing a mature FactorIX-containing polypeptide from a proprotein of Factor IX, or a conjugatethereof, refers to processing the proFIX or the conjugate thereof withPC5 to yield a mature Factor IX-containing polypeptide.

As used herein, a conjugate refers to any two or more entities bound toone another by any physicochemical means, including, but not limited to,hydrophobic interaction, covalent interaction, hydrogen bondinteraction, ionic interaction, and any combination thereof. Thus in oneembodiment a conjugate of the invention refers to any two or moreentities bound to one another by covalent interaction. For example, inone embodiment a conjugate is a fusion protein. In one embodiment aconjugate of the invention refers to any two or more entities bound toone another by noncovalent interaction.

The method according to this aspect of the invention involves contactingthe proFIX or the conjugate thereof with an effective amount offunctional PC5 polypeptide. Contacting refers to bringing the variousentities into intimate physical contact, e.g., so as to permit PC5 toprocess the proFIX or the conjugate thereof. An effective amount, asused herein, refers to an amount that is sufficient to achieve a desiredbiological effect, e.g., processing of the proFIX or the conjugatethereof by PC5.

In one embodiment the method according to this aspect of the inventioncan be practiced using an isolated proprotein of Factor IX, or aconjugate thereof, and an isolated PC5 polypeptide. For example, anisolated proprotein of Factor IX derived from one source can becontacted with an isolated PC5 polypeptide derived from another source,under conditions that allow processing of the proFIX or fusion proteinthereof by the functional PC5 polypeptide. Conditions that allowprocessing of the proFIX or fusion protein thereof by the functional PC5polypeptide include the presence of a source of vitamin K, gammacarboxylase, and additional components and conditions suitable for gammacarboxylase enzymatic activity.

In one embodiment according to this aspect of the invention, the matureFactor IX-containing polypeptide is a FIX-Fc monomer-dimer hybrid, suchas is disclosed in co-owned U.S. patent application Ser. No. 10/841,250,published as US 2005/0032174, the entire content of which isincorporated herein by reference. In this embodiment the proFIXconjugate corresponds to a proFIX-Fc monomer-dimer hybrid.

Also provided is a mature FIX-Fc monomer-dimer hybrid produced accordingto the method of this aspect of the invention.

As used herein, PEGylated proFIX refers to a conjugate formed betweenproFIX and at least one polyethylene glycol (PEG) molecule. PEG iscommercially available in a large variety of molecular weights andaverage molecular weight ranges. Typical examples of PEG averagemolecular weight ranges include, but are not limited to, 200, 300, 400,600, 1000, 1300-1600, 1450, 2000, 3000, 3000-3750, 3350, 3000-7000,3500-4500, 5000-7000, 7000-9000, 8000, 10000, 8500-11500, 16000-24000,35000, and 40000. These average molecular weights are provided merely asexamples and are not meant to be limiting in any way.

In one embodiment, the peptide of the invention may be PEGylated toinclude mono- or poly- (e.g., 2-4) PEG moieties. PEGylation may becarried out by any of the PEGylation reactions known in the art. Methodsfor preparing a PEGylated protein product will generally include (a)reacting a polypeptide with polyethylene glycol (such as a reactiveester or aldehyde derivative of PEG) under conditions whereby thepeptide of the invention becomes attached to one or more PEG groups; and(b) obtaining the reaction product(s). In general, the optimal reactionconditions for the reactions will be determined case by case based onknown parameters and the desired result.

There are a number of PEG attachment methods available to those skilledin the art, for example, EP 0 401 384; Malik F et al. (1992) ExpHematol. 20:1028-35; Francis (1992) Focus on Growth Factors 3(2):4-10;EP 0 154 316; EP 0 401 384; WO 92/16221; and WO 95/34326.

The step of PEGylation as described for the proteins of the inventionmay be carried out via an acylation reaction or an alkylation reactionwith a reactive polyethylene glycol molecule. Thus, protein productsaccording to the present invention include PEGylated proteins whereinthe PEG group(s) is (are) attached via acyl or alkyl groups. Suchproducts may be mono-PEGylated or poly-PEGylated (for example, thosecontaining 2-6 or 2-5 PEG groups). The PEG groups are generally attachedto the protein at the α- or ε-amino groups of amino acids, but it isalso contemplated that the PEG groups can be attached to any amino groupattached to the protein that is sufficiently reactive to become attachedto a PEG group under suitable reaction conditions.

PEGylation by acylation generally involves reacting an active esterderivative of polyethylene glycol with a peptide of the invention. Foracylation reactions, the polymer(s) selected typically have a singlereactive ester group. Any known or subsequently discovered reactive PEGmolecule may be used to carry out the PEGylation reaction. An example ofa suitable activated PEG ester is PEG esterified to N-hydroxysuccinimide(NHS). As used herein, acylation is contemplated to include, withoutlimitation, the following types of linkages between the therapeuticprotein and a polymer such as PEG: amide, carbamate, urethane, and thelike. See, for example, Charnow S M et al. (1994) Bioconjug Chem.5:133-40. Reaction conditions may be selected from any of those known inthe PEGylation art or those subsequently developed, but should avoidconditions such as temperature, solvent, and pH that would inactivatethe polypeptide to be modified.

PEGylation by acylation will generally result in a poly-PEGylatedprotein. The connecting linkage may be an amide. The resulting productmay be substantially only (e.g., >95%) mono-, di- or tri-PEGylated.However, some species with higher degrees of PEGylation may be formed inamounts depending on the specific reaction conditions used. If desired,more purified PEGylated species may be separated from the mixture(particularly unreacted species) by standard purification techniques,including among others, dialysis, salting-out, ultrafiltration,ion-exchange chromatography, gel filtration chromatography andelectrophoresis.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with a polypeptide in the presence of a reducingagent. For the reductive alkylation reaction, the polymer(s) selectedshould have a single reactive aldehyde group. An exemplary reactive PEGaldehyde is polyethylene glycol propionaldehyde, which is water stable,or mono C1-C10 alkoxy or aryloxy derivatives thereof. See, for example,U.S. Pat. No. 5,252,714.

Recombinantly produced FIX can be isolated from cells, culture media,and other products, for use in vitro and in vivo, including clinicaluse.

When proteins are produced according to the methods of the invention,they can occur in a mixture of molecules such as other proteins orprotein fragments. For example, the FIX-Fc monomer-dimer hybridsgenerally are expressed in a mixture of products which also includeFIX-Fc dimers and Fc dimers. The invention thus can involve methods ofisolating any of the proteins described supra from a mixture containingthe proteins. In particular, in one embodiment the invention can involvemethods for isolating a proFIX-Fc monomer-dimer hybrid from a mixture ofproteins including the monomer-dimer hybrid, a dimer, and at least aportion of an immunoglobulin constant region, e.g., an Fc fragment. Inone embodiment the invention can involve methods for isolating aproFIX-Fc homodimer from a mixture of proteins including themonomer-dimer hybrid, a dimer, and at least a portion of animmunoglobulin constant region, e.g., an Fc.

In one embodiment protein product is secreted into the media. Media isseparated from the cells, concentrated, filtered, and then passed overtwo or three affinity columns: a protein A column and one or two anionexchange columns.

In a typical example cells are separated from medium by centrifugationin disposable bottles. The separated medium is then filtered through0.8/0.2 μM PALL AcroPak filters, and concentrated 5-7 fold with aMillipore ProFlux M12 tangential flow filtration system, Pellicon 2cassettes, 10 K, B10-A, 0.5 m². Retentate is filtered using 0.8/0.2 μmPALL AcroPak filter into sterile plastic bags.

Protein A column (MabSelect, Amersham) is equilibrated with PBS, pH 7.4.The column is loaded with 10-15 mg protein/mL of resin at a flow rate of150-200 cm/hour and washed with PBS followed 3-5 column volumes of PBSplus 0.9 M NaCl. Prior to elution the conductivity is lowered with PBS.The column is then eluted with 25 mM sodium citrate/150 mM NaCl, pH 3.4,and fractions are neutralized with 2M Tris to a final pH of 7. Resultingeluate contains all Fc-containing species.

Eluate from the Protein A column are loaded onto a DEAE column(Fractogel, EMD) and the column is equilibrated with 25 mM Tris/150 mMNaCl, pH 7.5. After washing the loaded and equilibrated column with 3-5column volumes of 25 mM Tris/350 mM ammonium acetate, pH 7.5, the columnis eluted with 25 mM Tris/600 mM ammonium acetate, pH 7.5. The eluatecontains FIX-Fc monomer-dimer hybrid, free of FIX-Fc dimer and Fcfragments.

Although the purity of FIX-Fc monomer-dimer hybrid is already about 98percent following DEAE chromatography, as a further step to enrichclotting activity of the isolated FIX-Fc monomer-dimer hybrid, a secondion exchange chromatography step can be used. Eluate from the previousstep is diluted 1:4 with 25 mM Tris/150 mM NaCl, pH 7.5, and loaded ontoa Q Sepharose FF column (Amersham) at 7-10 mg/ml. After washing thecolumn with equilibration buffer, the column is eluted with 7 mMCaCl₂/150 mM NaCl/25 mM Tris, pH 7.5. Eluted FIX-Fc monomer-dimer hybridis collected until the UV signal falls to about 20 percent of themaximum absorbance at 280 nm. Less active FIX-Fc monomer-dimer hybridcan be stripped off the column with higher concentration of either CaCl₂(e.g., 10 mM) or ammonium acetate (e.g. 600 mM). Alternative andadditional methods of purification and enrichment are known in thefield, e.g., U.S. Pat. Nos. 4,981,952 and 5,714,583.

Using peptide mapping, it is possible to show propeptide is completelyprocessed from FIX-Fc monomer-dimer hybrid by PC5. Tryptic digest (LysC,ArgC) of FIX-Fc monomer-dimer hybrid generates a marker for propeptide(propeptide indicator peptide, “PIP”) if FIX propeptide is not fullyprocessed. The FIX propeptide has an amino acid sequenceTVFLDHENANKILNRPKR (SEQ ID NO:17), of which the sequence TVFLDHENANK(SEQ ID NO:18) represents the propeptide indicator peptide (PIP). PIP ispresent in FIX-Fc monomer-dimer hybrid transfected without processingenzyme (PC5), and it is absent in FIX-Fc monomer-dimer hybridtransfected with processing enzyme (PC5). PIP is also present in FIX-Fcmonomer-dimer hybrid transfected with PC7, and absent in FIX alonetransfected with PACE. In contrast, a reference peptide (“K23”corresponding to amino acid residues 395-400 of SEQ ID NO:3), having thesequence YGIYTK (SEQ ID NO:19), is present in FIX-Fc monomer-dimerhybrid transfected either with or without processing enzyme (PC5).

The activity of mature, activated FIX and FIX-containing polypeptides ofthe invention can be measured using a standard activated partialthromboplastin time (aPTT) assay. This assay is widely used as aclinical assay for Factor IX clotting activity. To perform the assay, atest sample is preincubated in FIX-deficient plasma with phospholipidsand an activator such as ellagic acid or silica, and then clot formationis induced by addition of CaCl₂. The time to clot formation is measured,e.g., with an MLA Electra 1600 clinical instrument, and compared to aWorld Health Organization (WHO) Factor IX standard.

A particular advantage of FIX-Fc fusion proteins and conjugates over FIXalone is their extended half-life in vivo. The extended half-lifepermits a radically reduced dosing requirement compared to FIX alone.For example, in one experiment HEK 293-derived FIX-Fc monomer-dimerhybrid was found to have a terminal half-life of 30.0 hours followingintravenous injection into normal rats. By comparison, the terminalhalf-life of BeneFIX® was found to be 5 hours following intravenousinjection into normal rats. In a separate set of experiments in normaldogs, the terminal half-lives of FIX-Fc monomer-dimer hybrid andBeneFIX® were found to be 36 hours and 12-14 hours, respectively,following intravenous administration. Surprisingly, the half-life ofFIX-Fc dimer in this set of experiments in dogs was found to be 22hours. In all these experiments, pharmacokinetic measurements wereperformed using enzyme-linked immunosorbent assay (ELISA) specific forFIX.

In addition to the ELISA-based results showing extended in vivohalf-life of FIX-Fc monomer-dimer hybrid, functional activity of FIX-Fcmonomer-dimer hybrid was also shown to be extended by measuring aPTT inFIX-deficient mice. Following a single intravenous dose of FIX-Fcmonomer-dimer hybrid (217 IU/kg body weight), the aPTT activity inplasma was shown to decay with a half-life of 47 hours in FIX-deficientmice.

The invention relates in part to a method of treating a subject having ahemostatic disorder comprising administering a therapeutically effectiveamount of at least one FIX polypeptide produced according to one or moremethods of the invention. In particular, the fusion proteins andconjugates of the invention can be used to treat or prevent a hemostaticdisorder associated with FIX deficiency, by promoting the formation of afibrin clot. A hemostatic disorder that may be treated by administrationof the fusion protein or conjugate of the invention includes, but is notlimited to, hemophilia B.

A FIX protein or conjugate of the invention can be used prophylacticallyto treat a subject with a hemostatic disorder. Alternatively or inaddition, a FIX fusion protein or conjugate of the invention of theinvention can be used to treat an acute bleeding episode in a subjectwith a hemostatic disorder. In one embodiment, the hemostatic disorderis the result of an inherited deficiency in Factor IX. In anotherembodiment the hemostatic disorder can be an acquired disorder. Theacquired disorder can result from an underlying secondary disease orcondition. The secondary disease or condition can be, for example andwithout limitation, liver disease, disseminated intravascularcoagulation (DIC), sepsis or infection, a cancer, an autoimmune disease,pregnancy, advanced age, or from medication to treat an underlyingsecondary disorder (e.g., cancer chemotherapy).

The FIX proteins and FIX conjugates of the invention can be administeredvia any suitable route of administration, including intravenously,subcutaneously, intramuscularly, or via any mucosal surface, e.g.,orally, sublingually, buccally, sublingually, nasally, rectally,vaginally or via pulmonary route. The FIX proteins and conjugates of theinvention can optionally be implanted within or linked to a biopolymersolid support that allows for the slow release of the FIX protein orconjugate to the desired site.

FIX-FcRn binding partner fusion proteins and other FcRn bindingpartner-containing conjugates in particular are well suited for deliveryto any epithelial surface that expresses FcRn. Such epithelial surfacesinclude but are not limited to oral, gastric, intestinal, intrabiliary,intranasal, and pulmonary including in particular large airways.

The dose of the FIX protein or conjugate of the invention will varydepending on the subject and upon the particular route of administrationused. Dosages can range from 0.1 to 100,000 μg/kg body weight. In oneembodiment, the dosing range is 0.1-2,000 μg/kg. The protein orconjugate can be administered continuously or at specific timedintervals. In vitro assays may be employed to determine optimal doseranges and/or schedules for administration. Additionally, effectivedoses may be extrapolated from dose-response curves obtained from animalmodels.

For clinical use the FIX proteins and conjugates of the invention can beformulated with any suitable pharmaceutical carrier. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences by E. W. Martin. Examples of excipients caninclude starch, glucose, lactose, sucrose, gelatin, malt, rice, flour,chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodiumchloride, dried skim milk, glycerol, propylene glycol, water, ethanol,and the like. The composition can also contain pH buffering reagents,and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take theform of tablets or capsules prepared by conventional means. Thecomposition can also be prepared as a liquid for example a syrup or asuspension. The liquid can include suspending agents (e.g., sorbitolsyrup, cellulose derivatives or hydrogenated edible fats), emulsifyingagents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil,oily esters, ethyl alcohol, or fractionated vegetable oils), andpreservatives (e.g., methyl- or propyl-p-hydroxybenzoates or sorbicacid). The preparations optionally can also include flavoring, coloring,and sweetening agents. Alternatively, the composition can be presentedas a dry (e.g., lyophilized) product for constitution with water oranother suitable vehicle.

For buccal and sublingual administration the composition may take theform of tablets, lozenges, or fast-dissolving films according toconventional protocols.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray from a pressurized pack or nebulizer (e.g., in PBS), with asuitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The pharmaceutical composition can be formulated for parenteraladministration (e.g., intravenous or intramuscular) by bolus injectionor infusion. Formulations for injection or infusion can be presented inunit dosage form, e.g., in ampoules or in multidose containers with anadded preservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form(e.g., lyophilized) for constitution with a suitable vehicle, e.g.,pyrogen-free water.

The pharmaceutical composition can also be formulated for rectaladministration as a suppository or retention enema, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

The FIX protein or conjugate of the invention can be used to treat asubject with a hemostatic disease or condition in combination with atleast one other known agent to treat said disease or condition. In oneembodiment, the invention relates to a method of treating a subject witha hemostatic disorder comprising administering a therapeuticallyeffective amount of at least one FIX protein or conjugate of theinvention, in combination with a source of at least one other clottingfactor or agent that promotes hemostasis. Said source in one embodimentis a preparation of plasma, e.g., fresh frozen plasma. Said otherclotting factor or agent that promotes hemostasis can be any therapeuticwith demonstrated clotting activity. As an example, but not as alimitation, the clotting factor or hemostatic agent can include FactorV, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII,Factor XII, prothrombin, fibrinogen, or activated forms of any of thepreceding. The clotting factor of hemostatic agent can also includeanti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamicacid.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting.

EXAMPLES Example 1 Cloning of Fc-Expressing Constructs

The coding sequence for the constant region of IgG1 (EU # 221-447; theFc region) was obtained by polymerase chain reaction (PCR) amplificationfrom a leukocyte cDNA library (Clontech, CA) using the followingoligonucleotide primers:

rcFc-F: (SEQ ID NO: 20) 5′- GCTGCGGTCGACAAAACTCACACATGCCCACCGTGCCCAGCTC-CGGAACTCCTGGGCGGACCGTCAGTC -3′ rcFc-R: (SEQ ID NO: 21)5′- ATTGGAATTCTCATTTACCCGGAGACAGGGAGAGGC- 3′

The forward primer rcFc-F adds three amino acids (AAV) and a Sailcloning site before the beginning of the Fc region, and alsoincorporates a BspEI restriction site at amino acids 231-233 and anRsrII restriction site at amino acids 236-238 using the degeneracy ofthe genetic code to preserve the correct amino acid sequence (EUnumbering). The reverse primer rcFc-R adds an EcoRI cloning site afterthe stop codon of the Fc. A 25 μl PCR reaction was carried out with 25pmol of each primer using Expand™ High Fidelity System (BoehringerMannheim, Indianapolis, Ind.) according to the manufacturer's standardprotocol in a MJ Thermocycler using the following cycles: 94° C. 2minutes; 30 cycles of (94° C. 30 seconds, 58° C. 30 seconds, 72° C. 45seconds); 72° C. 10 minutes. The expected sized band (˜696 bp) was gelpurified with a Gel Extraction kit (Qiagen, Valencia, Calif.), andcloned into pGEM T-Easy (Promega, Madison, Wis.) to produce anintermediate plasmid pSYN-Fc-001 (pGEM T-Easy/Fc).

The mouse Igκ signal sequence was added to the Fc CDS using thefollowing primers:

re-Igκ sig seq-F: (SEQ ID NO: 22)5′- TTTAAGCTTGCCGCCACCATGGAGACAGACACACTCCTGCTA-TGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACAAAACTC- ACACATGCCCACCG -3′Fc-noXma-GS-R: (SEQ ID NO: 23) 5′- GGTCAGCTCATCGCGGGATGGG -3′Fc-noXma-GS-F: (SEQ ID NO: 24) 5′- CCCATCCCGCGATGAGCTGACC -3′

The rc-Igκ signal sequence-F (rc-Igκ sig seq-F) primer adds a HindIIIrestriction site to the 5′ end of the molecule, followed by a Kozaksequence (GCCGCCACC; SEQ ID NO:25) followed by the signal sequence fromthe mouse Igκ light chain, directly abutted to the beginning of the Fcsequence (EU# 221). The Fc-noXma-GS-F and Fc-noXma-GS-R primers removethe internal XmaI site from the Fc coding sequence, using the degeneracyof the genetic code to preserve the correct amino acid sequence. Two 25μl PCR reactions were carried out with 25 pmol of either rc-Igκ signalsequence-F and Fc-noXma-GS-R or Fc-noXma-GS-F and rcFc-R using Expand™High Fidelity System (Boehringer Mannheim, Indianapolis, Ind.) accordingto the manufacturer's standard protocol in a MJ Thermocycler. The firstreaction was carried out with 500 ng of leukocyte cDNA library (BDBiosciences Clontech, Palo Alto, Calif.) as a template using thefollowing cycles: 94° C. 2 minutes; 30 cycles of (94° C. 30 seconds, 55°C. 30 seconds, 72° C. 45 seconds); 72° C. 10 minutes. The secondreaction was carried out with 500 ng of pSYN-Fc-001 (above) as atemplate using the following cycles: 94° C. 2 minutes; 16 cycles of (94°C. 30 seconds, 58° C. 30 seconds, 72° C. 45 seconds); 72° C. 10 minutes.The expected sized bands (˜495 and 299 bp, respectively) were gelpurified with a Gel Extraction kit (Qiagen, Valencia, Calif.), thencombined in a PCR reaction with 25 pmol of rc-Igκ signal sequence-F andrcFc-R primers and run as before, annealing at 58° C. and continuing for16 cycles. The expected sized band (˜772 bp) was gel purified with a GelExtraction kit (Qiagen, Valencia, Calif.) and cloned into pGEM T-Easy(Promega, Madison, Wis.) to produce an intermediate plasmid pSYN-Fc-007(pGEM T-Easy/Igκ sig seq-Fc). The entire Igκ signal sequence-Fc cassettewas then subcloned using the HindIII and EcoRI sites into pcDNA3.1(Invitrogen, Carlsbad, Calif.) mammalian expression vector to generatepSYN-Fc-015 (pcDNA3/Igκ sig seq-Fc).

The nucleic acid sequence for the insert in pSYN-Fc-015 is provided asSEQ ID NO:63, in which the signal sequence (first 60 nucleotides) isshown in italics.

SEQ ID NO: 63 atggagacag acacactcct gctatgggta ctgctgctctgggttccagg ttccactggt gacaaaactc acacatgcccaccgtgccca gctccggaac tgctgggcgg accgtcagtcttcctcttcc ccccaaaacc caaggacacc ctcatgatctcccggacccc tgaggtcaca tgcgtggtgg tggacgtgagccacgaagac cctgaggtca agttcaactg gtacgtggacggcgtggagg tgcataatgc caagacaaag ccgcgggaggagcagtacaa cagcacgtac cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaatggcaa ggagtacaagtgcaaggtct ccaacaaagc cctcccagcc cccatcgagaaaaccatctc caaagccaaa gggcagcccc gagaaccacaggtgtacacc ctgcccccat cccgcgatga gctgaccaagaaccaggtca gcctgacctg cctggtcaaa ggcttctatcccagcgacat cgccgtggag tgggagagca atgggcagccggagaacaac tacaagacca cgcctcccgt gttggactccgacggctcct tcttcctcta cagcaagctc accgtggacaagagcaggtg gcagcagggg aacgtcttct catgctccgtgatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a

For secretion of the Fc region, a heterologous signal peptide was used,specifically the mouse Igκ light chain signal sequence, as found inGenBank accession no. AB050084, Mus musculus VL2C mRNA for anti-A/dTantibody. In the Fc region, the nucleotide sequence was modified toincorporate restriction endonuclease sites while preserving the codingsequence. Specifically, the codons for A231, P232, and E233 (EUnumbering) were modified from GCA CCT GAA in the nucleotide sequence forthe Fc region of IgG1 in Genbank accession no. Y14735 to GCT CCG GAA atnucleotides 93 and 96 in order to incorporate a BspEI restriction sitewhile preserving the amino acid code. Also, the codons for G236, G237,and P238 (EU numbering) were modified from GGG GGA CCG to GGC GGA CCG atnucleotide 108 in order to incorporate a RsrII restriction site whilepreserving the amino acid code. Additionally, there was a noncodingdifference at Leu 234 from CTC to CTG at nucleotide 102, a noncodingdifference at R465 from CGG to CGC at nucleotide 465, and a noncodingdifference at L640 from CTG to TTG at nucleotide 592.

The amino acid sequence for the translated product encoded by SEQ IDNO:63 is provided as SEQ ID NO:64, where the signal peptide (first 20residues) is shown in italics.

SEQ ID NO: 64METDTLLLWV LLLWVPGSTG DKTHTCPPCP APELLGGPSV FLEPPKPKDT LMISRTPEVT 60CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK 120CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE 180WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS 240LSLSPGK 247

Example 2 Cloning of FIX-Fc-Expressing Constructs

The following primers were used to generate the various FIX-Fcexpression constructs (regions that annealed to the initial template areindicated in bold).

NatFIX-F: (SEQ ID NO: 26) 5′- TTACTGCAGAAGGTTATGCAGCGCGTGAACATG -3′F9-R-ZTM: (SEQ ID NO: 27) 5′- AGTGAGCTTTGTTTTTTCCTTAATCC -3′FIXaddXba-F: (SEQ ID NO: 28) 5′- CAAGGGAATCTAGAGAGAGAATGTATGGAAGAAAA-GTG -3′ FIXaddXba-R: (SEQ ID NO: 29)5′- ACATTCTCTCTCTAGATTCCCTTGAACAAACTCTTCC -3′ pEDF1-F: (SEQ ID NO: 30)5′- ATGACATCCACTTTGCCTTTCTCT -3′ fcclv-R: (SEQ ID NO: 31)5′- ATAGAAGCCTTTGACCAGGC -3′ FIX-Fc delta-F: (SEQ ID NO: 32)5′- AAAAACAAAGCTCACTGACAAAACTCACACATG- CCCACC -3′ FIX-Fc delta-R:(SEQ ID NO: 33) 5′- GTGTGAGTTTTGTCAGTGAGCTTTGTTTTTTCCTT- AATCCAG -3′IgkFc-NotI-F: (SEQ ID NO: 34)5′- ATGCGGCCGCGCCGCCACCATGGAGACAGACACACTC -3′ Fc-Xho-R: (SEQ ID NO: 35)5′- ATCTCGAGTCATTTACCCGGAGACAG -3′ FIXa5: (SEQ ID NO: 36)5′-GTCAAAGCTTCGCGACGTACGGCCGCCACCATGCAGCGCGTG- AACATGATC -3′ FIXa3:(SEQ ID NO: 37) 5′- CTGTGATGTTCCCACAGTACTTACCAACCTGCGTG -3′ FIXb5:(SEQ ID NO: 38) 5′- AGTACTGTGGGAACATCACAG -3′ FIXb3: (SEQ ID NO: 39)5′- TGACTCTAGATTCCCTTGAACAAACTCTTCCAA -3′

Construction of FIX-Fc Expression Plasmids

Construction of the FIX-Fc expression plasmids began with reversetranscriptase-polymerase chain reaction (RT-PCR) of the FIX codingsequence, followed by the generation of a number of differentintermediate plasmids used for expression during the early phases ofresearch before the final three plasmids were created: pSYN-FIX-021,pSYN-FIX-027, and pSYN-FIX-030. The final FIX-Fc constructs were allmade such that the FIX coding region was directly fused to the Fc codingsequence, with no intervening linker.

The native Factor IX (FIX) sequence (GenBank accession no. NM_(—)000133)was obtained by RT-PCR from human adult liver mRNA using the primersNatFIX-F and F9-R-ZTM with the Invitrogen Superscript RT-PCR withPlatinum Tag kit according to the manufacturer's standard protocol. Thecycle used was 30 min at 50° C. for the reverse transcription, followedby denaturing at 94° C. for 2 min and 35 cycles of (94° C. 30 sec, 55°C. 30 sec, 72° C. 1 min), followed by 10 min extension at 72° C. andthen storage at 4° C. The resulting PCR fragment was subcloned into thevector pGEM-T-Easy to generate pSYN-FIX-001. The entire sequence wasthen amplified using an extended version of these two primers to addrestriction sites (5′ BsiWI), Kozak sequence and a linker, and furtheramplified in three PCR reactions to add the Fc sequence and subcloned tocreate pSYN-FIX-002, for expressing FIX-Fc N297A in a different vectorsystem (pEE12.4/FIX-Fc N297A). Other sequences outside of the FIX codingsequence were subcloned into pSYN-FIX-002 to create pSYN-FIX-003(pEE12.4-6.4/FIX-Fc N297A/PACE) and pSYN-FIX-004 (pEE12.4-6.4/FIX-FcN297A/KEX2), before the FIX sequence was subcloned into a differentexpression system, pSYN-FIX-011 (pED.dC/FIX-Fc N297A).

The XbaI site was added to the FIX sequence in a manner similar to thatin which the XmaI site was removed from Fc, above. Briefly, the internalprimers FIXaddXba-F and FIXaddXba-R were used to add the XbaI site whilepreserving the amino acid sequence. Two 25 μl PCR reactions were carriedout with 50 pmol of either pEDFI-F and FIXaddXba-R or FIXaddXba-F andfcc1v-R using Expand™ High Fidelity System according to themanufacturer's standard protocol in a MJ Thermocycler. Both reactionswere carried out with 500 ng of pSYN-FIX-011 as a template using thefollowing cycles: 94° C. 2 min; 14 cycles of (94° C. 30 sec, 48° C. 30sec, 72° C. 2 min); 72° C. 10 min. Bands of the expected sizes (˜290 and1698 bp, respectively) were excised from an agarose gel and DNA purifiedwith a Gel Extraction kit, then combined in a PCR reaction with 50 pmolof pEDFl-F and fcc1v-R primers and run as before, annealing at 48° C.and continuing for 14 cycles. The plasmid pSYN-FIX-011 was used as thetemplate, and the primers pEDfl-F and fcc1v-R external to the PstI (5′)and Sail (3′) restriction sites were used in the same type of three PCRreactions to generate the identical FIX amino acid sequence whileincorporating the XbaI restriction site. The resulting 1965 bp fragmentwas digested with PstI/SalI, and subcloned back into the same sites inpSYN-FIX-011 to generate pSYN-FIX-013 (pED.dC/FIX-Fc N297A w/XbaI site).The Fc N297A sequence was subsequently cut out and replaced with thewildtype sequence to generate pSYN-FIX-016 (pED.dC/FIX-Fc w/XbaI site).

The sequence for the final FIX-Fc coding region (without any linker) wasgenerated in a similar manner. Briefly, the internal primers FIX-Fcdelta-F and FIX-Fc delta-R, which anneal to either the Fc or FIXsequence with a corresponding FIX or Fc overhang, were used to removethe linker region. Two 25 μl PCR reactions were carried out with 10 pmolof either pEDF1-F and FIX-Fc delta-R or FIX-Fc delta-F and rcFc-R usingExpand™ High Fidelity Polymerase similar to the manufacturer's standardprotocol, substituting in Failsafe™ Buffer E, in 10 μl reactions in theRapidcycler. Both reactions were carried out with 500 ng of pSYN-FIX-016(pED.dC/FIX-Fc w/XbaI) as a template using the following cycles: 94° C.1 min; 14 cycles of (94° C., 0 sec, 52° C., 0 sec, 72° C. 90 sec, slope6.0); 72° C. 10 min. The expected sized bands (˜1496 and 710 bp,respectively) were excised from an agarose gel and DNA purified with theEppendorf Perfectprep Gel Cleanup Kit®, then combined in a PCR reactionwith 10 pmol of pEDF1-F and rcFc-R primers and run as before, annealingat 52° C. and continuing for 14 cycles. The resulting 2200 bp fragmentwas digested with XbaI/RsrII, and the resulting 1255 bp fragment wassubcloned back into the same sites in pSYN-FIX-016 to generatepSYN-FIX-020 (pED.dC/FIX-Δlinker-Fc w/XbaI site).

The FIX-Δlinker-Fc sequence was subsequently cut out and subcloned intothe EcoRI/HindIII sites of pcDNA4/myc-His C to generate pSYN-FIX-021(pcDNA4/FIX-Fc w/XbaI site). Note that although this vector can be usedto add the myc and His tags to the protein of interest, this plasmid wasconstructed in such a way to only produce the untagged FIX-Fc protein.

A nucleotide sequence for FIX-Fc in pSYN-FIX-021, including theprepropeptide, is provided as SEQ ID NO:65.

SEQ ID NO: 65 atgcagcgcg tgaacatgat catggcagaa tcaccaggcctcatcaccat ctgcctttta ggatatctac tcagtgctgaatgtacagtt tttcttgatc atgaaaacgc caacaaaattctgaatcggc caaagaggta taattcaggt aaattggaagagtttgttca agggaatcta gagagagaat gtatggaagaaaagtgtagt tttgaagaag cacgagaagt ttttgaaaacactgaaagaa caactgaatt ttggaagcag tatgttgatggagatcagtg tgagtccaat ccatgtttaa atggcggcagttgcaaggat gacattaatt cctatgaatg ttggtgtccctttggatttg aaggaaagaa ctgtgaatta gatgtaacatgtaacattaa gaatggcaga tgcgagcagt tttgtaaaaatagtgctgat aacaaggtgg tttgctcctg tactgagggatatcgacttg cagaaaacca gaagtcctgt gaaccagcagtgccatttcc atgtggaaga gtttctgttt cacaaacttctaagctcacc cgtgctgaga ctgtttttcc tgatgtggactatgtaaatt ctactgaagc tgaaaccatt ttggataacatcactcaaag cacccaatca tttaatgact tcactcgggttgttggtgga gaagatgcca aaccaggtca attcccttggcaggttgttt tgaatggtaa agttgatgca ttctgtggaggctctatcgt taatgaaaaa tggattgtaa ctgctgcccactgtgttgaa actggtgtta aaattacagt tgtcgcaggtgaacataata ttgaggagac agaacataca gagcaaaagcgaaatgtgat tcgaattatt cctcaccaca actacaatgcagctattaat aagtacaacc atgacattgc ccttctggaactggacgaac ccttagtgct aaacagctac gttacacctatttgcattgc tgacaaggaa tacacgaaca tcttcctcaaatttggatct ggctatgtaa gtggctgggg acatgtcttccacaaaggga gatcagcttt agttcttcag taccttagagttccacttgt tgaccgagcc acatgtcttc gatctacaaagttcaccatc tataacaaca tgttctgtgc tggcttccatgaaggaggta gagattcatg tcaaggagat agtgggggaccccatgttac tgaagtggaa gggaccagtt tcttaactggaattattagc tggggtgaag agtgtgcaat gaaaggcaaatatggaatat ataccaaggt gtcccggtat gtcaactggattaaggaaaa aacaaagctc actgacaaaa ctcacacatgcccaccgtgc ccagctccgg aactcctggg cggaccgtcagtcttcctct tccccccaaa acccaaggac accctcatgatctcccggac ccctgaggtc acatgcgtgg tggtggacgtgagccacgaa gaccctgagg tcaagttcaa ctggtacgtggacggcgtgg aggtgcataa tgccaagaca aagccgcgggaggagcagta caacagcacg taccgtgtgg tcagcgtcctcaccgtcctg caccaggact ggctgaatgg caaggagtacaagtgcaagg tctccaacaa agccctccca gcccccatcgagaaaaccat ctccaaagcc aaagggcagc cccgagaaccacaggtgtac accctgcccc catcccggga tgagctgaccaagaaccagg tcagcctgac ctgcctggtc aaaggcttctatcccagcga catcgccgtg gagtgggaga gcaatgggcagccggagaac aactacaaga ccacgcctcc cgtgttggactccgacggct ccttcttcct ctacagcaag ctcaccgtggacaagagcag gtggcagcag gggaacgtct tctcatgctccgtgatgcat gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaa

The nucleotide sequence in the Factor IX portion was modified from whatis present in GenBank to incorporate an XbaI site without changing theamino acid code. Specifically, the coding sequence in the N59, L60, andE61 region was changed from AAC CTT GAG to AAT CTA GAG at nucleotides177 and 180, which adds an XbaI restriction site. There is also anon-coding change at V447 from GTA to GTG at nucleotide 1341. In the Fcregion, the nucleotide sequence was modified to incorporate restrictionendonuclease sites while preserving the coding sequence. Specifically,the codons in the region for A472, P473, and E474 (Fc EU numbering231-233) were modified from GCA CCT GAA in the nucleotide sequence forthe Fc region of IgG1 in GenBank accession no. Y14735 to OCT CCG GAA atnucleotides 1416 and 1419 in order to incorporate a BspEI restrictionsite while preserving the amino acid code. Also, the codons in theregion for G477, G478, and P479 (Fc EU numbering 236-238) were modifiedfrom GGG QUA CCG to GGC GGA CCG at nucleotide 1431 in order toincorporate a RsrII restriction site while preserving the amino acidcode. Finally, there is a noncoding difference at L640 from CTG to TTG.

The FIX-Fc amino acid sequence encoded by SEQ ID NO:65 is provided asSEQ ID NO:66, wherein the signal sequence (28 residues) is shown initalics and the propeptide (18 residues) is shown in bold.

SEQ ID NO: 66 MQRVNMIMAE SPGLITICLL GYLLSAECTV FLDHENANKI LNRPKRYNSG KLEEFVQGNL 60ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN PCLNGGSCKD DINSYECWCP 120FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD NKVVCSCTEG YRLAENQKSC EPAVPFPCGR 180VSVSQTSKLT RAETVFPDVD YVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW 240QVVLNGKVDA FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII 300PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS GYVSGWGRVF 360HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH EGGRDSCQGD SGGPHVTEVE 420GTSFLTGIIS WGEECAMKGK YGIYTKVSRY VNWIKEKTKL TDKTHTCPPC PAPELLGGPS 480VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST 540YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 600KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 660GNVFSCSVMH EALHNHYTQK SLSLSPGK 688

Construction of a Single Plasmid for the Coexpression of FIX-Fc and Fc

For construction of the single plasmid for the expression of FIX-Fc andFc, different restriction sites were added to the mouse Igκ signalpeptide-Fc coding sequence using the primers IgκFc-NotI-F and Fc-Xho-R.The template for this PCR reaction was pSYN-Fc-011 (pGEM T-Easy/Igκsignal sequence −Fc without BspEI, RsrII sites). A 10 μl PCR reactionwas set up with 100 ng template and 50 pmol of each primer using Expand™High Fidelity polymerase supplemented with Failsafe™ buffer E in theRapid Cycler. The PCR reaction was carried out using the followingcycles: 94° C. for 1 min; 15 cycles of (94° C. for 0 min, 48° C. for 0min and 72° C. for 1 min) followed by a final extension of 72° C. for 10min. The PCR product was run on an agarose gel, and the expectedfragment of ˜700 bp was excised and DNA purified using a QIAquick gelextraction kit. The PCR product was cloned into pCR2.1-Topo. The mouseIgκ signal peptide-Fc coding sequence was then cloned into pBudCE4.1using the NotI/XhoI restriction sites.

The nucleotide sequence for the Fc region in pBudCE4.1 (as well as inpSYN-FIX-027 and pSYN-FIX-030, below) is provided as SEQ ID NO:67.

SEQ ID NO: 67 atggagacag acacactcct gctatgggta ctgctgctctgggttccagg ttccactggt gacaaaactc acacatgcccaccgtgccca gcacctgaac tcctgggagg accgtcagtcttcctcttcc ccccaaaacc caaggacacc ctcatgatctcccggacccc tgaggtcaca tgcgtggtgg tggacgtgagccacgaagac cctgaggtca agttcaactg gtacgtggacggcgtggagg tgcataatgc caagacaaag ccgcgggaggagcagtacaa cagcacgtac cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaatggcaa ggagtacaagtgcaaggtct ccaacaaagc cctcccagcc cccatcgagaaaaccatctc caaagccaaa gggcagcccc gagaaccacaggtgtacacc ctgcccccat cccgcgatga gctgaccaagaaccaggtca gcctgacctg cctggtcaaa ggcttctatcccagcgacat cgccgtggag tgggagagca atgggcagccggagaacaac tacaagacca cgcctcccgt gttggactccgacggctcct tcttcctcta cagcaagctc accgtggacaagagcaggtg gcagcagggg aacgtcttct catgctccgtgatgcatgag gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a

The same mouse Igκ signal sequence used in pSYN-Fc-015 was utilized forthis construct. In the Fc region, the nucleotide sequence was modifiedto incorporate a restriction endonuclease site while preserving thecoding sequence. In the Fc region there are three noncoding differencescompared to the sequence or the Fc region of IgG1 in GenBank accessionno. Y14735: G236 (EU numbering) was modified from GGG to GGA atnucleotide 108, R465 (EU numbering) was modified from CGG to CGC atnucleotide 465, and a noncoding difference at L640 (EU numbering) wasmodified from from CTG to TTG at nucleotide 592.

The resulting amino acid sequence is identical to that of pSYN-Fc-015(SEQ ID NO:64).

FIX-Fc was then cloned into pBudCE4.1 that contained the IgκFc sequence.FIX-Fc was excised from pSYN-FIX-020 (above) using the enzymes HindIIIand EcoRI and was cloned into the same sites in pBudCE4.1/IgκFc. Thefinal plasmid containing FIX-Fc downstream of the CMV promoter and IgκFcdownstream of the EF1α promoter in pBudCE4.1 was named pSYN-FIX-027.

Construction of a Single Plasmid for the Coexpression of FIX-Fc (with aTruncated FIX Intron I) and Fc

An additional FIX-Fc-expression construct was generated which included atruncated form of the FIX intron I (GenBank accession no. NC_(—)000023),which had previously been shown to increase the level of FIX expressiondue to functional splicing sequences present in the precursor mRNA.Kurachi S et al. (1995) J Biol Chem. 270:5276-81. The truncated portionof the FIX intron I was obtained by PCR initially in two pieces thatwere then assembled together in a third PCR reaction as follows. Two 50μl PCR reactions were carried out with 45 and 90 pmol of either theprimers FIXa5 and FIXa3 or FIXb5 and FIXb3 using Expand™ High FidelityPolymerase according to the manufacturer's standard protocol in the MJThermocycler. Both reactions were carried out with human genomic DNA asa template using the following cycles: 94° C. 3 min; 16 cycles of (94°C. 30 sec, 58° C. 30 sec, 72° C. 90 sec); 72° C. 13.5 min. The 141 and157 bp sequences of the 5′ and 3′ end region, respectively, of FIXintron I were obtained and joined by PCR in a second set of PCRreactions that were carried out as before, using a mixture of theseproducts as the new templates and the primers FIXa5 and FIXb3. Thisfragment was initially cloned into the intermediate vector pCR2.1 TOPOto generate pSYN-FIX-028 (pcR2.1/FIX mini intron1). The HindIII/XbaIfragment containing the FIX mini intron within the context of the FIXCDS (“minigene”) was then subcloned into pSYN-FIX-027 to generatepSYN-FIX-030 (pBUD/FIX-Fc mini intron 1/Fc).

A nucleotide sequence for FIX-Fc in pSYN-FIX-030, including the intron,is provided as SEQ ID NO:68. This nucleotide sequence matches that ofthe pSYN-FIX-021 construct (see SEQ ID NO:65), apart from the insertionin pSYN-FIX-030 of the 299 bp truncated intron approximately between thesignal peptide coding sequence and the propeptide coding sequence.

SEQ ID NO: 68 atgcagcgcg tgaacatgat catggcagaa tcaccaggcctcatcaccat ctgcctttta ggatatctac tcagtgctgaatgtacaggt ttgtttcctt ttttaaaata cattgagtatgcttgccttt tagatataga aatatctgat gctgtcttcttcactaaatt ttgattacat gatttgacag caatattgaagagtctaaca gccagcacgc aggttggtaa gtactgtgggaacatcacag attttggctc catgccctaa agagaaattggctttcagat tatttggatt aaaaacaaag actttcttaagagatgtaaa attttcatga tgttttcttt tttgctaaaactaaagaatt attcttttac atttcagttt ttcttgatcatgaaaacgcc aacaaaattc tgaatcggcc aaagaggtataattcaggta aattggaaga gtttgttcaa gggaatctagagagagaatg tatggaagaa aagtgtagtt ttgaagaagcacgagaagtt tttgaaaaca ctgaaagaac aactgaattttggaagcagt atgttgatgg agatcagtgt gagtccaatccatgtttaaa tggcggcagt tgcaaggatg acattaattcctatgaatgt tggtgtccct ttggatttga aggaaagaactgtgaattag atgtaacatg taacattaag aatggcagatgcgagcagtt ttgtaaaaat agtgctgata acaaggtggtttgctcctgt actgagggat atcgacttgc agaaaaccagaagtcctgtg aaccagcagt gccatttcca tgtggaagagtttctgtttc acaaacttct aagctcaccc gtgctgagactgtttttcct gatgtggact atgtaaattc tactgaagctgaaaccattt tggataacat cactcaaagc acccaatcatttaatgactt cactcgggtt gttggtggag aagatgccaaaccaggtcaa ttcccttggc aggttgtttt gaatggtaaagttgatgcat tctgtggagg ctctatcgtt aatgaaaaatggattgtaac tgctgcccac tgtgttgaaa ctggtgttaaaattacagtt gtcgcaggtg aacataatat tgaggagacagaacatacag agcaaaagcg aaatgtgatt cgaattattcctcaccacaa ctacaatgca gctattaata agtacaaccatgacattgcc cttctggaac tggacgaacc cttagtgctaaacagctacg ttacacctat ttgcattgct gacaaggaatacacgaacat cttcctcaaa tttggatctg gctatgtaagtggctgggga agagtcttcc acaaagggag atcagctttagttcttcagt accttagagt tccacttgtt gaccgagccacatgtcttcg atctacaaag ttcaccatct ataacaacatgttctgtgct ggcttccatg aaggaggtag agattcatgtcaaggagata gtgggggacc ccatgttact gaagtggaagggaccagttt cttaactgga attattagct ggggtgaagagtgtgcaatg aaaggcaaat atggaatata taccaaggtgtcccggtatg tcaactggat taaggaaaaa acaaagctcactgacaaaac tcacacatgc ccaccgtgcc cagetccggaactcctgggc ggaccgtcag tcttcctctt ccccccaaaacccaaggaca ccctcatgat ctcccggacc cctgaggtcacatgcgtggt ggtggacgtg agccacgaag accctgaggtcaagttcaac tggtacgtgg acggcgtgga ggtgcataatgccaagacaa agccgcggga ggagcagtac aacagcacgtaccgtgtggt cagcgtcctc accgtcctgc accaggactggctgaatggc aaggagtaca agtgcaaggt ctccaacaaagccctcccag cccccatcga gaaaaccatc tccaaagccaaagggcagcc ccgagaacca caggtgtaca ccctgcccccatcccgggat gagctgacca agaaccaggt cagcctgacctgcctggtca aaggcttcta tcccagcgac atcgccgtggagtgggagag caatgggcag ccggagaaca actacaagaccacgcctccc gtgttggact ccgacggctc cttcttcctctacagcaagc tcaccgtgga caagagcagg tggcagcaggggaacgtctt ctcatgctcc gtgatgcatg aggctctgcacaaccactac acgcagaaga gcctctccct gtctccgggt aaa

The resulting FIX-Fc amino acid sequence is identical to that ofpSYN-FIX-021 and pSYN-FIX-027.

Example 3 Cloning of PC5

The coding sequence for human PC5 was obtained by RT-PCR. The followingprimers were used (areas that anneal to the cDNA are indicated in bold):

PC5-KpnI-F: (SEQ ID NO: 40) 5′- ATCTACACCATCTCCATCAGCAGC -3′ PC5 NotI-R:(SEQ ID NO: 41) 5′- AAGGCGGCCGCTCAGCCTTGAAATGTACATGTTTTGC -3′ PC5-UTR-F:(SEQ ID NO: 42) 5′- AGCGAGGGAGCAGCGAGG -3′ PC5-HindIII-R:(SEQ ID NO: 43) 5′- GGTAGTTGACATGGCGGTTGG -3′ PC5-Af12-F:(SEQ ID NO: 44) 5′- CAGCGACTTAAGCCACCATGGGCTGGGGGAGCCG -3′ PC5-KpnI-R:(SEQ ID NO: 45) 5′- GTAGGTTGTGGCCAGCGTGG -3′

Coding sequence for human PC5 (GenBank accession no. NM_(—)006200) wasobtained in two pieces. The 3′ ˜1750 bp were obtained using the primersPC5-KpnI-F and PC5-NotI-R with the Invitrogen Superscript RT-PCR withPlatinum Taq kit according to the manufacturer's standard protocol, fromhuman liver mRNA. The cycle used for the reverse transcription was 30min at 50° C. followed by denaturing at 94° C. for 2 min and 35 cyclesof 94° C. for 15 sec, 54° C. for 30 sec, 72° C. for 3 min, followed by10 min extension at 72° C. and then storage at 4° C. This produced afragment from the internal KpnI site in the PC5 coding sequence throughthe stop codon, with a NotI site added at the 3 end. This fragment wasthen cloned into pCR2.1 TOPO according to manufacturer's protocol togenerate pSYN-PC5-001 (pCR2.1/PC5 (KpnI-NotI)). This fragment was thensubcloned into pcDNA3.1/hygro using the KpnI and NotI restriction sitesto generate pSYN-PC5-002 (pcDNA3.1/hygro/PC5 (KpnI-NotI)).

The 5′ ˜1100 bp of PC5 was obtained in two steps. It was first amplifiedby RT-PCR using the primers PC5-UTR-F and PC5-HindIII-R to amplify a˜1520 bp fragment from human liver mRNA, using similar conditions asabove, with an annealing temperature of 57° C. These primers havecomplete homology to the native PC5 sequence, in the untranslated 5′sequence and sequence 3′ from the internal unique HindIII site,respectively. Note that this HindIII site is not present in the finalconstruct due to a silent nucleotide substitution. This DNA fragment wasthen gel purified and used as a template for a second PCR reaction withPC5-Afl2-F, which adds an AflII cloning site followed by a Kozaksequence to the N-terminal coding sequence at the 5′ end, andPC5-KpnI-R, which anneals 3′ to the internal unique KpnI site, togenerate an ˜1100 bp fragment. The reaction was carried out with theExpand™ High Fidelity System according to the manufacturer's standardprotocol in a MJ Thermocycler using the following cycles: 94° C. 2 min;14 cycles of (94° C. 30 sec, 57° C. 30 sec, 72° C. 2 min), followed by72° C. 10 min. This fragment was then subcloned into pSYN-PC5-002 usingthe AflII and KpnI restriction sites to generate pSYN-PC5-003(pcDNA3.1/hygro/PC5).

The nucleotide sequence encoding PC5 in pSYN-PC5-003 has the followingsequence (SEQ ID NO: 61):

SEQ ID NO: 61 atgggctggg ggagccgctg ctgctgcccg ggacgtttggacctgctgtg cgtgctggcg ctgctcgggg gctgcctgctccccgtgtgt cggacgcgcg tctacaccaa ccactgggcagtcaaaatcg ccgggggctt cccggaggcc aaccgtatcgccagcaagta cggattcatc aacataggac agataggggccctgaaggac tactaccact tctaccatag caggacgattaaaaggtcag ttatctcgag cagagggacc cacagtttcatttcaatgga accaaaggtg gaatggatcc aacagcaagtggtaaaaaag cggacaaaga gggattatga cttcagtcgtgcccagtcta cctatttcaa tgatcccaag tggcccagtatgtggtatat gcactgcagt gacaatacac atccctgccagtctgacatg aatatcgaag gagcctggaa gagaggctacacgggaaaga acattgtggt cactatcctg gatgacggaattgagagaac ccatccagat ctgatgcaaa actacgatgctctggcaagt tgcgacgtga atgggaatga cttggacccaatgcctcgtt atgatgcaag caacgagaac aagcatgggactcgctgtgc tggagaagtg gcagccgctg caaacaattcgcactgcaca gtcggaattg ctttcaacgc caagatcggaggagtgcgaa tgctggacgg agatgtcacg gacatggttgaagcaaaatc agttagcttc aacccccagc acgtgcacatttacagcgcc agctggggcc cggatgatga tggcaagactgtggacggac cagcccccct cacccggcaa gcctttgaaaacggcgttag aatggggcgg agaggcctcg gctctgtgtttgtttgggca tctggaaatg gtggaaggag caaagaccactgctcctgtg atggctacac caacagcatc tacaccatctccatcagcag cactgcagaa agcggaaaga aaccttggtacctggaagag tgttcatcca cgctggccac aacctacagcagcggggagt cctacgataa gaaaatcatc actacagatctgaggcagcg ttgcacggac aaccacactg ggacgtcagcctcagccccc atggctgcag gcatcattgc gctggccctggaagccaatc cgtttctgac ctggagagac gtacagcatgttattgtcag gacttcccgt gcgggacatt tgaacgctaatgactggaaa accaatgctg ctggttttaa ggtgagccatctttatggat ttggactgat ggacgcagaa gccatggtgatggaggcaga gaagtggacc accgttcccc ggcagcacgtgtgtgtggag agcacagacc gacaaatcaa gacaatccgccctaacagtg cagtgcgctc catctacaaa gcctcaggctgctcagataa ccccaaccgc catgtcaact acctggagcacgtcgttgtg cgcatcacca tcacccaccc caggagaggagacctggcca tctacctgac ctcgccctct ggaactaggtctcagctttt ggccaacagg ctatttgatc actccatggaaggattcaaa aactgggagt tcatgaccat tcattgctggggagaaagag ctgctggtga ctgggtcctt gaagtttatgatactccctc tcagctaagg aactttaaga ctccaggtaaattgaaagaa tggtctttgg tcctctacgg cacctccgtgcagccatatt caccaaccaa tgaatttccg aaagtggaacggttccgcta tagccgagtt gaagacccca cagacgactatggcacagag gattatgcag gtccctgcga ccctgagtgcagtgaggttg gctgtgacgg gccaggacca gaccactgcaatgactgttt gcactactac tacaagctga aaaacaataccaggatctgt gtctccagct gcccccctgg ccactaccacgccgacaaga agcgctgcag gaagtgtgcc cccaactgtgagtcctgctt tgggagccat ggtgaccaat gcatgtcctgcaaatatgga tactttctga atgaagaaac caacagctgtgttactcact gccctgatgg gtcatatcag gataccaagaaaaatctttg ccggaaatgc agtgaaaact gcaagacatgtactgaattc cataactgta cagaatgtag ggatgggttaagcctgcagg gatcccggtg ctctgtctcc tgtgaagatggacggtattt caacggccag gactgccagc cctgccaccgcttctgcgcc acttgtgctg gggcaggagc tgatgggtgcattaactgca cagagggcta cttcatggag gatgggagatgcgtgcagag ctgtagtatc agctattact ttgaccactcttcagagaat ggatacaaat cctgcaaaaa atgtgatatcagttgtttga cgtgcaatgg cccaggattc aagaactgtacaagctgccc tagtgggtat ctcttagact taggaatgtgtcaaatggga gccatttgca aggatgcaac ggaagagtcctgggcggaag gaggcttctg tatgcttgtg aaaaagaacaatctgtgcca acggaaggtt cttcaacaac tttgctgcaa aacatgtaca tttcaaggc

SEQ ID NO:61 contains substitutions from the GenBank sequence that donot affect the amino acid coding sequence. Specifically, the nucleotideat position 399 (corresponding to position 876 of GenBank accession no.NM_(—)006200) is a T instead of a C, but preserves the amino acid Ser133 (corresponding to amino acid numbering in GenBank accession no.NP_(—)006191); nucleotide position 1473 (GenBank position 1950) is a Cinstead of a T, but preserves the amino acid Ala 491; and nucleotideposition 1485 (GenBank position 1962) is an A instead of a G, butpreserves the amino acid Ser 496. Note that the nucleotide change atposition 1473 eliminates a HindIII restriction site.

Example 4 Cloning of PACE-SOL

The coding sequence for human PACE was obtained by RT-PCR. The followingprimers were used (areas that anneal to the cDNA are indicated in bold):

PACE-F1: (SEQ ID NO: 46) 5′- GGTAAGCTTGCCATGGAGCTGAGGCCCTGGTTGC -3′PACE-R1: (SEQ ID NO: 47) 5′- GTTTTCAATCTCTAGGACCCACTCGCC -3′ PACE-F2:(SEQ ID NO: 48) 5′- GCCAGGCCACATGACTACTCCGC -3′ PACE-R2: (SEQ ID NO: 49)5′- GGTGAATTCTCACTCAGGCAGGTGTGAGGGCAGC -3′

The primer PACE-F1 adds a HindIII site to the 5′ end of the PACEsequence beginning with 3 nucleotides before the start codon, while theprimer PACE-R2 adds a stop codon after amino acid 715, which occurs atthe end of the extracellular domain of PACE, as well as adding an EcoRIsite to the 3′ end of the stop codon. The PACE-R1 and PACE-F2 primersanneal on the 3′ and 5′ sides of an internal BamHI site, respectively.Two RT-PCR reactions were then set up using 25 pmol each of the primerpairs of PACE-F1/R1 or PACE-F2/R2 with 20 ng of adult human liver RNA(Clontech; Palo Alto, Calif.) in a 50 μl RT-PCR reaction using theSuperScript™ One-Step RT-PCR with PLATINUM® Taq system (Invitrogen,Carlsbad, Calif.) according to manufacturer's protocol. The reaction wascarried out in a MJ Thermocycler using the following cycles: 50° C. 30minutes; 94° C. 2 minutes; 30 cycles of (94° C. 30 seconds, 58° C. 30seconds, 72° C. 2 minutes), followed by 72° C. 10 minutes. Thesefragments were each ligated into the vector pGEM T-Easy (Promega,Madison, Wis.) and sequenced fully. The F2-R2 fragment was thensubcloned into pcDNA6 V5/His (Invitrogen, Carlsbad, Calif.) using theBamHI/EcoRI sites, and then the F1-R1 fragment was cloned into thisconstruct using the HindIII/BamHI sites. The final plasmid, pcDNA6-PACE,produces a soluble form of PACE (amino acids 1-715), as thetransmembrane region has been deleted. The sequence of PACE inpcDNA6-PACE is essentially as described in Harrison S et al. (1998)Semin Hematol 35(2 Suppl 2):4-10.

Example 5 Cloning of Kex2-SOL

Coding sequence for the yeast endoprotease, KEX2, was obtained by RT-PCRfrom Saccharomyces cerevisiae polyA+ mRNA (BD Clontech, cat # 6999-1)using the following primers (areas that anneal to the cDNA are indicatedin bold):

KEX2-F: (SEQ ID NO: 50) 5′- GCGCTAGCCGTACGGCCGCCACCATGAAAGTGAGGAAATA-TATTACTTTATGC -3′ KEX2-BglII-F: (SEQ ID NO: 51)5′- GCTATTGATCACAAAGATCTACATCCTCC -3′ KEX2-BglII-R: (SEQ ID NO: 52)5′- GGAGGATGTAGATCTTTGTGATCAATAGC -3′ KEX2-675-R: (SEQ ID NO: 53)5′- GCGAATTCCGGTCCGTCATTGCCTAGGGCTCGAGAG- TTTTTTAGGAGTGTTTGGATCAG -3′

These primers were used to obtain coding sequence for KEX2 (amino acids1-675), the yeast homolog to PACE, in two pieces in a manner similar tothat used for PACE-SOL, Example 4 above; similarly, the transmembraneregion was removed to generate the soluble form of the protein.

Example 6 Cloning of PC7-SOL

Coding sequence for PC7 was obtained by RT-PCR from human adult livermRNA using the following primers (areas that anneal to the cDNA areindicated in bold):

PC7-BamMut-F: (SEQ ID NO: 54) 5′- GCATGGACTCCGATCCCAACG -3′PC7-BamMut-R: (SEQ ID NO: 55) 5′- CGTTGGGATCGGAGTCCATGC -3′ PC7-F:(SEQ ID NO: 56) 5′- GGTAAGCTTGCCGCCACCATGCCGAAGGGGAGGCAGAAAG -3′PC7-SOL-R: (SEQ ID NO: 57) 5′- TTTGAATTCTCAGTTGGGGGTGATGGTGTAACC -3′PC7-Xma-F: (SEQ ID NO: 58) 5′- GGCACCTGAATAACCGACGG -3′ PC7-Xma-R:(SEQ ID NO: 59) 5′- CGTCACGTTGATGTCCCTGC -3′

These primers were used to obtain coding sequence for PC7 (amino acids1-663) in three pieces in a manner similar to that used for PACE-SOL,Example 4 above; similarly, the transmembrane region was removed togenerate the soluble form of the protein.

Example 7 Generation of Propeptide Antibody

A peptide with the sequence TVFLDHENANKILNRPKRC (SYN1117; SEQ ID NO:60)corresponding to the 18 amino acid propeptide sequence of FIX, with aCys residue added at the C terminus for conjugation to Keyhole LimpetHemocyanin (KLH) was synthesized. This peptide was conjugated to KLH,two New Zealand white rabbits were immunized, and antisera collected.

Peptide SYN 1117 was conjugated to Ultralink® Iodoacetyl Gel (Pierce,Rockford, Ill.) according to manufacturer's protocol. The gel-linkedpeptide was subsequently packed into a column and the antibody was thenaffinity purified over the immobilized propeptide column by gravityflow. 10 ml of antisera was filtered through a 0.2 μm sterile syringefilter to remove particulate matter. The antiserum was then applied tothe column in 1.5 ml fractions, each of which was allowed to bind theresin for 1 hour. The column was washed with PBS, and the purifiedantibody eluted in 100 mM glycine, pH 2.8, then neutralized with 1 MTris, pH 9.0. Antibody-containing fractions (as determined by A280) werepooled and dialyzed into PBS and 0.2% sodium azide was added forstorage.

Example 8 Transfection of Lines HEK 293 Transfections

HEK 293H cells were adapted to adherent culture using serum-containingmedium. Suspension cells were transferred to a T75 flask containingDulbecco's modified Eagle medium (DMEM) (high glucose) supplemented with10% fetal bovine serum (FBS) and 0.1 mM non-essential amino acids andgrown as stationary cultures at 37° C./5% CO₂. Adherent HEK 293H cellswere subcultured every three to four days. Subculturing was performed bydislodging cells from the bottom of the flask by gently pipetting themedium up and down.

10-cm² Dishes

10-cm² dishes were seeded with 2.5×10⁶ cells in 10 ml adherent growthmedium one day prior to transfection. Transfections were carried outusing calcium phosphate Profection transfection reagent (Promega,Madison, Wis.). Three hours before transfection, medium was replacedwith 10 ml fresh growth medium. For each transfection 500 μl of 2×HEPESbuffered saline (2×HBS) was added to a sterile tube. In a second steriletube, 20 μg of DNA (either 9 μg FIX-021/9 μg Fc-015/2 μg PC5-003, or 9μg FIX-027/9 μg Fc-015/2 μg PC5-003, or 9 μg FIX-030/9 μg Fc-015/2 μgPC5-003) was mixed with 62 μl of 2M CaCl₂ and the total volume increasedto 500 μl with sterile water. The DNA/CaCl₂ mix was added dropwise tothe 2×HBS while vortexing. The DNA mixture was incubated for 30 minutesat room temperature and then vortexed again briefly before addingdropwise to the cells. Cells were incubated with the DNA mixture for 16hours at 37° C./5% CO₂ before removing the transfection solution,washing the cells with Hanks balanced salt solution (HBSS) and thenadding 10 ml fresh growth medium to the cells.

48 to 72 hours after transfection, cells were removed from 10-cm² dishesby pipetting up and down and were each split into 10-cm² dishes in 5 mlfresh growth medium or split into 96-well plates with 100 μl growthmedium per well. 24 hours after plating, selection was initiated byadding an equal volume of growth medium containing the appropriateantibiotic to give the final concentrations of hygromycin at 200 μg/mlor Zeocin, Geneticin, and Hygromycin at 25, 75, and 25 μg/ml. Oncecolonies of antibiotic-resistant cells began to form, cells wereisolated from 10-cm² dishes with cloning rings or expanded from 96-wellplates to 24-well plates, then to T25 flasks and T75 flasks beforeadapting the cell lines back to serum-free suspension culture. As cellswere transferred from 96-well plates and 10-cm² dishes to 24-wellplates, the antibiotic concentration was decreased by one half and cellswere then maintained in this reduced antibiotic medium.

6-Well Dishes

6-well dishes were seeded with 5×10⁵ cells in 2 ml adherent growthmedium one day prior to transfection. Transfections were carried outusing calcium phosphate Profection transfection reagent (Promega,Madison, Wis.). Three hours before transfection, medium was replacedwith 2 ml fresh growth medium. For each transfection 83 μl of 2×HBS wasadded to a sterile tube. In a second sterile tube, 4 μg of DNA (3.6 μgFIX-027/0.4 μg PC5-003) was mixed with 10 μl of 2M CaCl₂ and the totalvolume increased to 83 μl with sterile water. The DNA/CaCl₂ mix wasadded dropwise to the 2×HBS while vortexing. The DNA mixture wasincubated for 30 minutes at room temperature and then vortexed againbriefly before adding dropwise to the cells. Cells were incubated withthe DNA mixture for 16 hours at 37° C./5% CO₂ before removing thetransfection solution, washing the cells with HBSS and then adding 2 mlfresh growth medium to the cells.

40 to 72 hours after transfection, cells were removed from 6-well platesby pipetting up and down and transferring cells to T75 flasks. 24 hoursafter plating, selection was initiated by adding an equal volume ofgrowth medium containing hygromycin at a final concentration of 200μg/ml. Once a pool of stably selected cells was generated, cells werecounted and diluted to 1 cell/well in 96-well plates. As clonal celllines became confluent, cells were expanded from 96-well plates to24-well plates, T25 and T75 flasks and were then adapted back toserum-free suspension culture as described above.

All other HEK transfections were carried out in a similar manner,substituting in Kex2-SOL or PC7-SOL expressing plasmids for PC5. Forthese transfections, Lipofectamine™ 2000 (Invitrogen, Carlsbad, Calif.)transfection reagent was used according to manufacturer's protocolinstead of calcium phosphate.

CHO transfections were carried out in a similar manner, utilizingsimilar FIX-Fc expressing plasmids with the same processing enzymeexpression plasmids. In some cases, SuperFect (Qiagen, Valencia, Calif.)transfection reagent was used according to manufacturer's protocol inplace of calcium phosphate.

Example 9 Analysis of Transient and Stable Cell Lines Western Blotting

For analysis of all transient transfections, CHO stable transfections ofFIX-Fc with PC5, and HEK 293 stable transfections of FIX-Fc with PC7,conditioned media from the cells was subject to protein Aimmunoprecipitation. Briefly, cell culture supernatant was mixed withapproximately 40 μl of protein A-Sepharose 50% slurry and incubated at4° C. with rocking for 1 hour, then centrifuged to pellet the protein Abeads. Beads were resuspended in 1 ml of PBS or similar buffer, spundown, and buffer aspirated, and the process repeated 1-2 times. Thebeads were then resuspended with either reduced or non-reduced sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loadingdye, heated from 30 sec to 5 min, spun down, and the eluted protein inthe dye was loaded on SDS-PAGE gels and run according to standardprotocols. Gels were transferred to nitrocellulose membranes and Westernblots performed as described below.

For analysis of all other stable transfections, protein was firstpurified before being analyzed in Western blots. CHO produced FIX-Fcthat was transfected alone, with PACE, or PC7 was purified over proteinA, eluted, run on SDS-PAGE, transferred to nitrocellulose, and Westernblots performed. HEK produced FIX-Fc with PC5 was subjected tothree-column purification (see below) and analyzed in a similar manner.

Fc Western Blot

The antibody used for Fc Western blotting experiments was a goatanti-human IgG (Fc specific)-horseradish peroxidase conjugate (PierceImmunoPure® antibody, catalog # 31416). This antibody was diluted1:10,000 in PBS-T (PBS with 0.1% Tween-20) and incubated with themembrane for 1 hour at room temperature with gentle rocking. After three10 minute washes in PBS-T of approximately 20 ml each, chemiluminescentdetection was performed.

FIX Western Blot

For FIX Western blotting experiments, a goat anti-human FIX-horseradishperoxidase conjugate (Enzyme Research Laboratories, catalog#FIX-EIA-130D) was used. The antibody was diluted 1:1000 in PBS-T andincubated with the membrane for 1 hour at room temperature with gentlerocking. After three 10 minute washes of approximately 20 ml each, themembrane was ready for chemiluminescent detection.

FIX Propeptide Western Blot

For the propeptide Western blot, rabbit anti-FIX propeptide antibody(see Example 7) was diluted 1:10,000 in PBS-T and incubated with themembrane for 1 hour at room temperature with gentle rocking. Themembrane was subsequently placed into three 10-minute washes ofapproximately 20 ml each. The secondary (detection) antibody was a goatanti-rabbit IgG-horseradish peroxidase conjugate (Southern BiotechnologyAssociates, catalog #4010-05) diluted 1:20,000 in PBS-T. The secondaryantibody was incubated with the membrane for 30 minutes to 1 hr at roomtemperature, and then washed three times in approximately 20 ml of PBS-Tbuffer for 15 minutes each in preparation for chemiluminescentdetection.

Chemiluminescent Detection

Detection of all immunoblots was performed using the ECL Plus WesternBlotting Detection System (Amersham Biosciences catalog #RPN2132)according to the manufacturer's instructions. Visualization of thesignal was performed on a Storm 840 Phosphorimager (Molecular Devices).

Example 10 Summary of Propeptide Processing Experiments Western Blotting

Transient transfections in CHO cells initially indicated that whileFIX-Fc transfected alone retained the propeptide, cotransfection ofFIX-Fc with either PC5, PACE-SOL, PC7-SOL, or KEX2-SOL was sufficient tocompletely remove the propeptide. As shown in FIG. 4, FIX (top panels)and FIX propeptide (bottom panels) Western blots of transienttransfections in CHO cells initially indicated that FIX-Fc transfectedalone retained the propeptide (bands present on both FIX and propeptideWestern blots, lanes 1 and 5). In contrast, Western blots of transienttransfections in CHO cells initially indicated that cotransfection ofFIX-Fc with PC5 (lane 4), PACE-SOL (lanes 2 and 6), PC7-SOL (lanes 3 and7), or KEX2-SOL (lane 8) was sufficient to completely remove thepropeptide (bands present in FIX but not propeptide Western blots).Note, however, that cotransfection with KEX2-SOL (lane 8) resulted in anadditional, smaller FIX-containing band (top panel), likely due tocryptic cleavage by this more promiscuous enzyme (Rockwell et al. (2002)Chem. Rev. 102: 4525-48) at an additional site in FIX.

FIG. 5 demonstrates that in stably transfected CHO cell lines, PACE-SOLis capable of fully processing the propeptide, while PC7-SOL is not.Comparison was made between Protein A-purified material from a CHO cellline stably expressing FIX-Fc dimer, monomer, and Fc, cotransfected witheither PACE-SOL or PC7-SOL. Beginning with the cotransfection withPACE-SOL, lane 1 of the SDS-PAGE gel in panel A identifies all threespecies, lane 1 of the FIX Western in panel B confirms that the dimerand monomer bands contain FIX, and lane 1 of the propeptide Western inpanel C demonstrates (by the absence of bands) that the propeptide isfully processed by PACE. In contrast, with respect to cotransfectionwith PC7-SOL lane 2 of the SDS-PAGE gel in panel A again identifies allthree species, lane 2 of the FIX Western in panel B again confirms thatthe dimer and monomer bands contain FIX, but lane 2 of the propeptideWestern in panel C demonstrates (by presence of bands) that thepropeptide is still present in the FIX-Fc dimer and monomercotransfected with PC7-SOL.

FIG. 6 demonstrates that in stably transfected CHO cell lines, PC5 iscapable of fully processing the propeptide. In FIG. 6, lanes 1, 2, and 3show FIX Western (top panel) and propeptide Western (lower panel)results for protein A pulldowns from cell lines stably expressing FIX-Fcdimer, monomer, and Fc cotransfected with PC5. Lanes 1, 2 and 3correspond to stable cell lines amplified with 25, 50, and 100 nMmethotrexate, respectively. In the FIX Western, FIX-Fc dimer and monomerbands are indicated, and the Fc band does not react. The propeptideWestern confirmed that the dimer and monomer bands do not containpropeptide (no significant bands found). In FIG. 6, lanes 4 and 5 showFIX Western (top panel) and propeptide Western (lower panel) results forprotein A pulldowns from controls, CHO cell lines transfected witheither FIX-Fc alone (lane 4) or FIX-Fc together with PACE-SOL (lane 5).As shown in lane 5, FIX-Fc purified from CHO cell lines transfected withPACE-SOL (lane 5) is fully processed (note presence of strong bands inFIX Western, top panel, but only faint background bands in thepropeptide Western, bottom panel). In contrast, lane 4 shows that thepropeptide is still present in FIX-Fc purified from CHO cell lineswithout any cotransfected processing enzyme (note presence of strongbands in both FIX and propeptide Westerns, top and bottom panels,respectively).

In summary, examination of stably transfected CHO cell linesdemonstrated that only PACE-SOL (FIG. 5) and PC5 (FIG. 6) were able tocompletely process the propeptide, while FIX-Fc transfected alone (FIG.6) or with PC7-SOL (FIG. 5) produced proFIX-Fc.

The ability of these various enzymes to remove the propeptide was alsoexamined in HEK 293 cells. FIG. 7 shows that PC5, but not PC7-SOL,completely removed the propeptide from FIX-Fc derived from transientlytransfected HEK 293 cells. For the results shown in FIG. 7, HEK 293-Hcells were transiently transfected with FIX-Fc and Fc alone; FIX-Fc, Fcand PC5; or FIX-Fc, Fc, and PC7-SOL. The resulting conditioned media wassubject to protein A pulldowns, reducing SDS-PAGE, and Fc Westernblotting (left panel) or propeptide Western blotting (right panel). Asshown in FIG. 7, FIX-Fc protein derived from transfections without anyprocessing enzyme retained the propeptide (lanes 3, “−”), as did FIX-Fccotransfected with PC7-SOL (lanes 8-11, from transfections performed inquadruplicate), as can be seen from bands present in both Fc andpropeptide Westerns. In contrast, PC5 completely removed the propeptide(lanes 4-7 from transfections performed in quadruplicate), as indicatedby bands present only in the Fc, but not the propeptide, Western blots.As controls for these Westerns, FIX-Fc dimer purified from CHO cellstransfected alone (lanes 1) or with PACE-SOL (lanes 2) were analyzed.Propeptide was present in FIX-Fc transfected without any processingenzyme (bands in lanes 1 of both Westerns) but absent in FIX-Fccontransfected with PACE-SOL (lanes 2, band present in Fc Western, butonly faint background band in Propeptide Western).

FIG. 8 shows that KEX2-SOL was unable to process proFIX-Fc derived fromHEK 293 cells transiently transfected with FIX-Fc. Conditioned mediafrom HEK 293 cells transiently transfected with FIX-Fc, Fc, and KEX2-SOLwas subject to protein A pulldowns and either non-reducing SDS-PAGE andFc Western blotting (top panel) or reducing SDS-PAGE and propeptideWestern blotting (bottom panel). As shown in FIG. 8, FIX-Fccotransfected with KEX2-SOL is not processed, as can be seen by thepresence of propeptide in this material (lanes 3 and 4, bands present onboth blots). Lane 3 material has wildtype Fc in all three species(dimer, monomer, and Fc alone), while lane 4 material has N297A Fc. Ascontrols, FIX-Fc dimer purified from CHO cells transfected alone(lane 1) or with PACE-SOL (lane 2) were analyzed, confirming that thepropeptide was present in FIX-Fc transfected without any processingenzyme (bands in both Westerns, lane 1) but absent in FIX-Fccontransfected with PACE-SOL (lane 2, band present in Fc Western, butonly background band in propeptide Western).

In summary, in transiently transfected HEK 293 cells, only PC5demonstrated the ability to completely remove the propeptide (FIG. 7),while PC7-SOL (FIG. 7) and KEX2-SOL (FIG. 8) were unable to processproFIX-Fc, similar to FIX-Fc transfected without any processing enzyme(FIG. 7).

The ability of these enzymes to remove the propeptide was also examinedin stably transfected HEK 293 cells. FIG. 9 demonstrates that in stablytransfected cell lines, PC5 is capable of fully processing thepropeptide. Two lots of purified FIX-Fc monomer (see Example 11 forpurification process) from a HEK 293-H cell line stably expressingFIX-Fc dimer, monomer, and Fc cotransfected with PC5 were run induplicate (lanes 4a/b and 5a/b) on an SDS-PAGE gel under non-reducingconditions, then transferred to blots and probed with FIX (FIG. 9B) orpropeptide (FIG. 9C) antibodies. The gel was then stained after transferwith Gelcode Blue (Pierce, Rockford, Ill.) (FIG. 9A). Analysis of thefigures demonstrates that the propeptide was fully processed by PC5, asindicated by the absence of bands in panel C, propeptide Western, lanes4b and 5b. As can be seen from panel A, lanes 4a/b and 5a/b were allloaded with equal amounts of protein, confirmed to be FIX-Fc dimer andmonomer, as can be seen in panel B, lanes 4a and 5a. As controls,protein A-purified FIX-Fc dimer, monomer, and Fc from CHO cell linescotransfected with PACE-SOL (lanes 1a/b) or PC7-SOL (lanes 2a/b) wereanalyzed. These controls confirmed that the propeptide was present inthe material cotransfected with PC7 (bands present in panel C, lane 2b,as well as panel B, lane 2a), but not PACE-SOL (bands absent in panel C,lane 2b, but present in panel B, lane 2a).

FIG. 10 demonstrates that, in contrast to PC5, PC7-SOL was incapable ofprocessing the propeptide in stably transfected HEK 293 cell lines. Asshown in FIG. 10, media from HEK 293-H cells stably transfected eitherwith FIX-Fc and Fc alone, or with FIX-Fc, Fc, and PC7-SOL were subjectto protein A pulldowns and analyzed by reducing SDS-PAGE and Fc Westernblotting (top panel) or FIX propeptide Western blotting (bottom panel).The cells were transfected with the N297A version of FIX-Fc and Fc ineither equal ratios (lanes 3 and 5) or in a 1:8 ratio (lanes 4 and 6) ofFIX-Fc to Fc expression plasmids, respectively, either alone (lanes 3and 4) or with 1/10 the total DNA of PC7-SOL expression plasmid (lanes 5and 6). These analyses showed that the FIX-Fc protein retains thepropeptide whether stably transfected with (lanes 5 and 6) or without(lanes 3 and 4) PC7-SOL, as can be seen by bands in both the Fc Westernblot (top panel) and propeptide Western blot (bottom panel). As controlsfor these Westerns, FIX-Fc dimer purified from CHO cells transfectedalone (lane 1) or with PACE-SOL (lane 2) were analyzed, confirming thatthe propeptide was present on FIX-Fc transfected without any processingenzyme (bands in both Westerns, lane 1) but absent in FIX-Fccontransfected with PACE-SOL (lane 2, band present in Fc Western, butonly faint background band in propeptide Western).

In summary, stable HEK 293 cell lines appeared identical to thetransient transfections, in that PC5 was able to completely process thepropeptide (FIG. 9) while PC7 was not (FIG. 10).

Table 2 below summarizes the results of the different combinations ofprocessing enzymes and FIX-Fc proteins as assessed by Western blotting.

TABLE 2 Propeptide Processing Cell Transfection PC5 Kex2 PC7 PACE PACE 4CHO transient processes processes processes processes FIG. 4 (crypticFIG. 4 FIG. 4 cleavage) FIG. 4 stable processes not done does notprocess does not FIG. 6 fully process FIG. 5, 6 fully FIG. 5 process*HEK transient processes does not does not not done FIG. 7 fully processfully process FIG. 8 FIG. 7 stable processes not done does not not doneFIG. 9 fully process FIG. 10 *Wasley LC et al. (1993) J Biol Chem. 268:8458-65.

Example 11 Protein Purification Protein A Chromatography

A 5 cm×6 cm bed height column (117 ml volume, XK 5 column, Amersham) waspacked with MabSelect media according to manufacturer's specifications.The column was equilibrated with PBS and then loaded with theconcentrated media at 150 cm/h, providing a retention time of 2.4minutes. Following loading, the column was first washed with 3-4 volumesof PBS, then 3-4 volumes of PBS+0.9 M NaCl. Finally, the conductivitywas lowered with 3 volumes of PBS prior to elution. The bound proteineluted with 3-4 volumes of 25 mM Sodium Citrate+150 mM NaCl, pH 3.4.Following elution the column was stripped with 3 M Guanidine HCl. Theeluted material was neutralized with 2 M Tris base 8 ml per 100 ml ofeluate). The amount of protein eluted was estimated by measuring thetriplicate dilutions of the neutralized pool (1 in 10) in PBS. Theconcentration was determined using the equation

mg/ml=(absorbance 280−absorbance 320)/1.34

where 1.34 is the theoretical molar absorbance coefficient based on thenumber of tryptophans, tyrosines and disulfide bonds in FIX-Fc monomer.Gill et al. (1989) Analytical Biochem 182:319.

Anion Exchange Chromatography Using Fractogel DEAE

The neutralized Protein A eluate was diluted 1:1 with 25 mM Tris+150 mMNaCl, pH 7.5, and loaded on a 2.6 cm×7 cm bed height Fractogel DEAEcolumn (37 ml Volume, XK 2.6 column, Amersham). The Fractogel DEAEcolumn was packed according to the manufacturer's specifications, whichincluded 25% bed compression after packing by high flow.

The column was equilibrated with 6 column volumes (CV) of 25 mM Tris+150mM NaCl, pH 7.5, and loaded at ˜230 cm/h. The load was washed with 4 CVof equilibration buffer followed by 4 CV of 25 mM Tris+350 mM AmmoniumAcetate, pH 7.5, followed by 3 CV of equilibration buffer.

The FIX-Fc monomer eluted with 25 mM Tris+600 mM Ammonium Acetate, pH7.5 (˜5 CV). The column was then stripped with 5 volumes of 0.1 M SodiumHydroxide, followed by re-equilibration with 4 CV of 25 mM Tris+150 mMNaCl, pH 7.5.

Pseudoaffinity Chromatography Using Q Sepharose FF

Pseudoaffinity anion exchange chromatography involves CaCl₂ in elutingthe immobilized FIX-Fc from the column. The addition of CaCl₂ isbelieved to cause a conformational change in the FIX part of themolecule that causes it to elute from the Q Sepharose FF media. Yan S Bet al. (1996) J Mol Recognit. 9:211-8; Harrison S et al. (1998) SeminHematol 35(2 Suppl 2):4-10.

The DEAE eluate was diluted a total of 4-fold with 25 mM Tris+150 mMNaCl, pH 7.5. A 1.6 cm×11 cm bed height Q Seph FF column (22 ml volume,XK 1.6 column, Amersham) was equilibrated with 25 mM Tris+150 mM NaCl,pH 7.5 (9 CV). The column was then loaded at 200 cm/h and washed with 15CV of equilibration buffer. To ensure adequate binding of the FIX-Fcactive species, the DEAE load of new material is now diluted 1:4 with 25mM Tris+120 mM NaCl, pH 7.5, which now becomes the column equilibrationbuffer. The rest of the procedure remains the same.

The most active FIX-Fc monomer species eluted with 25 mM Tris+7 mMCaCl₂+150 mM NaCl, pH 7.5 (5 CV). Less active species eluted with 25 mMTris+10 mM CaCl₂+150 mM NaCl, pH 7.5 (5 CV), while least active FIX-Fcmaterial eluted with 25 mM Tris+600 mM Ammonium Acetate, pH 7.5.

The rFIX-Fc peak was collected from the beginning of the elution (UVsignal reaches ˜100 mAU) to when the UV signal reaches 20% of themaximum peak absorbance at the back end of the elution peak (˜30 mL).

The column was then stripped with 0.1 M Sodium Hydroxide (5 CV) and thenre-equilibrated with 25 mM Tris+150 mM NaCl, pH 7.5.

Buffer Exchange of Final Protein Preparation

The Q Seph FF elution fractions were buffer exchanged against PBS. Theprotein was injected in pre-wetted 125 ml Slide-A-lyzers (Pierce) andwas dialyzed to against two changes of PBS buffer, each time in excessof 200-fold PBS over protein solution.

Example 12 Analysis of Stable Cell Line and Peptide Mapping

For peptide mapping, tryptic digests were performed and the resultingpeptides analyzed by LC/MS to determine the presence of a fragment ofthe propeptide designated the propeptide indicator peptide, or PIP(TVFLDHENANK; SEQ ID NO:18), that would only be present in the case ofincomplete propeptide processing.

Approximately 100-200 μg of protein was dried down with a Speed Vac,then resuspended in 100-200 μl of digestion buffer (50 mM ammoniumbicarbonate) using an Eppendorf Thermal Mixer at 30° C., 800 rpm, for 30min. The sample was then reduced by adding 10 μl reducing agent (10 mMdithiothreitol in digest buffer) for 30 min at 56° C., 800 rpm on theThermal Mixer. The sample was allowed to cool, then 10 μl of alkylatingagent (55 mM iodoacetamide in digest buffer) was added and allowed toincubate for 30 mM at room temperature in the dark. Promega SequencingGrade Modified Trypsin at 0.1 μg/μl in 50 mM ammonium bicarbonate wasthen added at 100:1 to 200:1 substrate to enzyme weight ratio, thenincubated at 37° C., 800 rpm for 16 hours. The sample was dried downsomewhat with a Speed Vac as necessary to reduce volume, and the samplesstored at −20° C. until ready for analysis, as described below.

HPLC-UV Analysis—Materials and Equipment:

-   -   Agilent 1100 binary HPLC pump with thermostatted autosampler,        Diode Array Detector, and column heater    -   Phenomenex Jupiter HPLC Column, 250 mm×2.00 mm, C18, 300 Å, 5 μm        dp    -   Buffer A: 0.01% (v/v) Trifluoroacetic Acid, 0.05% (v/v) Formic        Acid. (in-house HPLC grade water, analytical grade reagents)    -   Buffer B: Buffer A in 85% HPLC grade Acetonitrile

HPLC-UV-MS Analysis—Materials and Equipment:

-   -   Agilent 1100 binary HPLC pump with thermostatted autosampler,        Diode Array Detector, and column heater    -   Thermo Finnigan Ion Trap Mass Spectrometer    -   Phenomenex Jupiter HPLC Column, 250 mm×2.00 mm, C18, 300 Å, 5 μm        dp    -   Buffer A: 0.01% (v/v) Trifluoroacetic Acid, 0.05% (v/v) Formic        Acid. (in-house HPLC grade water, analytical grade reagents)    -   Buffer B: Buffer A in 85% HPLC grade Acetonitrile

HPLC-UV Analysis—Procedure:

-   -   Gradient Program:

Time, min % Solvent B 0.00 5.6 25.00 16.8 50.00 21.3 71.00 29.0 91.0032.5 125.00 44.7 130.00 100 140.00 100 140.10 5.6 155.00 5.6

-   -   Flowrate: 0.250 ml/min;    -   Detection: Diode Array Detector, UV, 214 nm and 280 nm;    -   Column Temperature: 30° C.

HPLC-UV-MS Analysis—Procedure:

-   -   Gradient Program, Flowrate, Column Temperature, and UV Detection        as above.    -   MS Detection: A Thermo Finnigan LCQ Advantage Ion Trap Mass        Spectrometer connected in-line after the Diode Array Detector        and operated in the Electrospray Ionization (ESI) mode. Mass        spectrometric protocols varied depending on the goal of the        analysis, but were usually either “Triple Play” (sequential full        scan, zoom scan, and MSMS scan) for identification of peptides        or full scan only to produce mass chromatograms.

Example 13 Summary of Propeptide Processing Experiments Peptide Mapping

Western blotting results of stably transfected proteins were confirmedby peptide mapping. The propeptide indicator peptide (PIP) was detectedboth in the UV trace and selected ion trace in CHO FIX-Fc transfectedalone, but undetectable in purified FIX-Fc monomer cotransfected withPC5 (FIG. 11). ProFIX-Fc was also found in CHO FIX-Fc protein that wascontransfected with PC7, although possibly at lower levels as indicatedin the UV trace (FIG. 12). FIX alone produced in the presence ofPACE-SOL (BeneFIX®) appeared identical to FIX-Fc monomer produced withPC5 (FIG. 13), displaying no detectable PIP.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention. The entire contents of all of the references (includingliterature references, issued patents, published patent applications,and co-pending patent applications) cited throughout this applicationare hereby expressly incorporated by reference.

1-77. (canceled)
 78. An isolated host cell comprising a firstpolynucleotide and a second polynucleotide, wherein the firstpolynucleotide encodes a proprotein of Factor IX (proFIX) or a fusionprotein comprising said proFIX, and the second polynucleotide encodes afunctional PC5.
 79. The host cell of claim 78, wherein the functionalPC5 comprises SEQ ID NO:
 1. 80. The host cell of claim 78, wherein thefusion protein comprises proFIX fused to a heterologous polypeptide. 81.The host cell of claim 80, wherein the fusion protein further comprisesa linker connecting the proFIX and the heterologous polypeptide.
 82. Thehost cell of claim 81, wherein said linker is selected from the groupconsisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10,SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, and SEQ ID NO:
 16. 83. The host cell of claim 80, wherein theheterologous polypeptide is selected from the group consisting of anFcRn binding partner, albumin, and transferrin.
 84. The host cell ofclaim 83, wherein said FcRn binding partner comprises Fc.
 85. The hostcell of claim 84, wherein said Fc is dimerized to a second Fc, formingproFIX-Fc monomer-dimer hybrid.
 86. The host cell of claim 78, whereinthe proFIX or fusion protein comprising said proFIX is modified, whereinsaid modification is selected from the group consisting ofglycosylation, acetylation, phosphorylation, PEGylation, addition of alipid moiety, and addition of any organic or inorganic molecule.
 87. Thehost cell of claim 78, wherein the first polynucleotide and the secondpolynucleotide are in separate vectors.
 88. The host cell of claim 78,wherein the first polynucleotide and the second polynucleotide are in asingle vector.
 89. The host cell of claim 78, which is a prokaryoticcell.
 90. The host cell of claim 89, wherein the prokaryotic cell is E.coli.
 91. The host cell of claim 78, which is an eukaryotic cell. 92.The host cell of claim 91, wherein said cell is selected from the groupconsisting of a HEK293 cell, a CHO cell, a HeLa cell, and a BHK cell.93. A method of producing mature Factor IX or a fusion proteincomprising mature Factor IX comprising culturing the host cell of claim78 under conditions that allow expression of the proFIX or the fusionprotein comprising said proFIX and the functional PC5 such that, uponexpression, said PC5 cleaves proFIX or the fusion protein comprisingsaid proFIX.
 94. The method of claim 93, wherein said method increasesyield of the mature Factor IX or fusion protein comprising mature FactorIX compared to the yield of mature Factor IX or a fusion proteincomprising mature Factor IX produced under similar conditions withoutprocessing by the functional PC5.
 95. The method of claim 94, whereinthe functional PC5 comprises SEQ ID NO:
 1. 96. The method of claim 94,wherein the fusion protein comprises a proFIX fused to a heterologouspolypeptide.
 97. The method of claim 96, wherein the heterologouspolypeptide is selected from the group consisting of an FcRn bindingpartner, albumin, and transferrin.
 98. The method of claim 97, whereinsaid FcRn binding partner comprises Fc.
 99. The method of claim 98,wherein said Fc is dimerized to a second Fc, forming proFIX-Fcmonomer-dimer hybrid.
 100. The method of claim 94, wherein the proFIX orfusion protein comprising proFIX is modified, wherein said modificationis selected from the group consisting of glycosylation, acetylation,phosphorylation, PEGylation, addition of a lipid moiety, and addition ofany organic or inorganic molecule.